Use of certain drugs for treating nerve root injury

ABSTRACT

The present invention relates to a method and a pharmaceutical composition for treatment of nerve disorders comprising administration of a therapeutically effective dosage of at least two substances selected from the group consisting of TNF inhibitors, IL-1 inhibitors, IL-6 inhibitors, IL-8 inhibitors, FAS inhibitors, FAS ligand inhibitors, and IFN-gamma inhibitors. Preferably, at least one of the substances is a TNF inhibitor.

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/826,893 filed Apr. 6, 2001, which is acontinuation-in-part of U.S. patent application Ser. No. 09/743,852filed Jan. 17, 2001, which was a National Stage filing under 35 U.S.C. §371 of International Application No. PCT/SE99/01671 filed Sep. 23, 1999which was published in English on Apr. 6, 2000 and claims benefit ofSwedish Application Nos. 9803276-6 and 9803710-4 filed respectively onSep. 25, 1998 and Oct. 29, 1998. These applications are hereinincorporated by reference in their entirety for all purposes.

TECHNICAL FIELD

[0002] The present invention relates to a method for treating nervedisorders in a mammal or a vertebrate by administering at least twocytokine inhibitors, of which one is preferably a TNF inhibitor. Theinvention also relates to the use of at least two cytokine inhibitors,of which one is preferably a TNF inhibitor in the preparation ofpharmaceutical compositions for the treatment of nerve root injury.

[0003] The object of the present invention is to obtain an improvedpossibility to treat nerve disorders, such as nerve root injury inducedby disk herniation, which may turn up e.g. as a radiating pain in thearm or leg (sciatica), as low back pain or as whiplash associateddisorder, by blocking disk related cytokines.

BACKGROUND OF THE INVENTION

[0004] It is established that conditions such as sciatica and low backpain are due to activation and irritation of intraspinal nervousstructures by disk derived substances (Olmarker K, Rydevik B, NordborgC, Autologous nucleus pulposus induces neurophysiologic and histologicchanges in porcine cauda equina nerve roots, Spine 1993; 18(11):1425-32; Olmarker K, Larsson K, Tumor necrosis factor alpha andnucleus-pulposus-induced nerve root injury, Spine 1998; 23(23): 2538-44;Olmarker K, Rydevik B, Selective inhibition of tumor necrosisfactor-alpha prevents nucleus pulposus-induced thrombus formation,intraneural edema, and reduction of nerve conduction velocity: possibleimplications for future pharmacologic treatment strategies of sciatica,Spine 2001; 26(8) :863-9). One key substance for inducing suchirritation is Tumor Necrosis Factor alpha (TNF or TNF-alpha). TNF is aproinflammatory cytokine that may sensitize a nerve root in a way thatwhen it is simultaneously deformed mechanically, ectopic nerve may beelicited locally and interpreted by the brain as pain in thecorresponding dermatome. TNF may also induce a nutritional deficit inthe nerve root by increasing the vascular permeability leading tointraneural edema, and by initiating intravascular coagulation byactivation of adhesion molecules at the surface of the endothelial cells(Olmarker K, Rydevik B, Selective inhibition of tumor necrosisfactor-alpha prevents nucleus pulposus-induced thrombus formation,intraneural edema, and reduction of nerve conduction velocity: possibleimplications for future pharmacologic treatment strategies of sciatica,Spine 2001;26(8): 863-9). Both these mechanisms may subsequently lead toa reduced blood flow with a reduced supply of nutrients and eliminationof metabolic waist products. This reduction in nutrition may also inducesciatic pain per se. TNF may also induce low back pain due to localirritation of sensory nerve endings at the surface of the intervertebraldisk. This may occur when the nucleus pulposus herniates out into thespinal canal and TNF produced and released from the disk cells may reachthe nerve endings.

[0005] Disk herniation is a troublesome disorder, which can causepronounced pain and muscle dysfunction, and thereby loss of ability towork. A herniation may occur in any disk in the spine but herniations inthe lumbar and the cervical spine are most common. A disk herniation inthe cervical spine may induce radiating pain and muscle dysfunction inthe arm, which is generally referred to as cervical rhizopathy.Herniation in the lumbar spine may induce radiating pain and muscledysfunction in the leg. The radiating pain in the leg is generallyreferred to as sciatica. Disk herniation will cause trouble to a varyingdegree, and the pain may last for one or two months or in severe casesup to 6 months. The arm or leg pain that can occur as a result of diskherniation can be very intense and may thus affect the individualpatient's whole life situation during the sickness period.

[0006] U.S. Pat. No. 5,703,092 discloses the use of hydroxamic acidcompounds and carbocyclic acids as metalloproteinase and TNF inhibitors,for the treatment of arthritis and other related inflammatory diseases.No use of these compounds for the treatment of nerve root injuries isdisclosed or suggested.

[0007] U.S. Pat. No. 4,925,833 discloses the use of tetracyclines toenhance bone protein synthesis and treatment of osteoporosis.

[0008] U.S. Pat. No. 4,666,897 discloses inhibition of mammaliancollagenolytic enzymes by administering tetracyclines. Thecollagenolytic activity is manifested by excessive bone resorption,periodontal disease, rheumatoid arthritis, ulceration of cornea, orresorption of skin or other connective tissue collagen.

[0009] However, neither this nor U.S. Pat. No. 4,925,833 disclose nerveroot injury or the treatment thereof.

[0010] It has also been disclosed that selective inhibition may beefficient in reducing sciatic pain (Korhonen K, Karppinen J, MalmivaaraA, Paimela L, Kyllönen E, Lindgren K-A, et al. Treatment of sciaticawith infliximab, a monoclonal humanised chimaeric antibody against TNF.Trans. International Society for the Study of the Lumbar Spine 2002;Cleveland, Ohio, p. 14).

[0011] Low back pain affects approximately 80% of the population duringtheir lifetime in most countries. Except for being extremely common, itis also one of the most costly disorders for the society. In Swedenalone, low back pain was estimated to cost $320,000,000 in 1997. Themajor part of the cost relates to indirect costs such assick-compensation and reduced productivity, and only a minor part isrelated to direct costs such as medical care and pharmacologicalsubstances.

[0012] In a minority of the cases (5%), there may be a known cause forthe pain such as intra spinal tumors, rheumatic diseases, infections andmore. In these cases the treatment may be specifically aimed at thecause. However, in the majority of the cases of low back pain, the causeremains unknown. At present there is no direct way to treat low backpain with an unknown cause and existing treatment modalities only aim atsymptomatic relief.

[0013] Low back pain and sciatica

[0014] It is necessary to make a distinction between low back pain andone specific condition that is often linked to low back pain called“sciatica”. Sciatica refers to radiating pain into the leg according tothe dermatomal innervation area of a specific spinal nerve root. Thepain in sciatica is distinctly different from that of low back pain. Insciatica, the pain is sharp and intense, often described as“toothache-like”, and radiates down into the lower extremities, belowthe level of the knee. The experience of the pain is closely related tothe dermatomal innervation of one or more lumbar spinal nerve roots.Sciatica is also frequently related to neurological dysfunction in thatspecific nerve and may be seen as sensory dysfunction, reduced reflexesand reduced muscular strength. The sciatic pain thus seem to be aneuropathic pain, i.e. pain due to nerve injury, induced by sensitizedaxons in a spinal nerve root at the lumbar spinal level. The painexperienced by the patient at low back pain is more dull and isdiffusely located in the lower back. There is never any radiating paininto the leg.

[0015] Sciatica is the result of nerve injury, and the cause of sciaticahas an anatomical correlate. Since 1934, sciatica is intimately linkedto the presence of a herniated intervertebral disc. However, althoughmost patients with sciatica will display a herniated disc atradiological examination, it is surprising that approximately 30% of anadult population at the age of 40-50 years of age with no present orprevious sciatica also have disc herniations when assessed by magneticresonance tomography, so called “silent” disc herniations (Wiesel,Tsourmas et al. 1984; Boden, Davis et al. 1990; Boos, Rieder et al.1995; Boos, Dreier et al. 1997). The presence of silent disc herniationsis intriguing to the spine research community and seems to contradictthe relationship between disc herniations and sciatica.

[0016] Scientific knowledge of the pathophysiologic mechanisms behindlow back pain

[0017] It is well known that the outer part of the annulus fibrosus ofthe intervertebral disc and the posterior longitudinal ligament areinnervated by C-fibers. Although there are no nerve fibers in the deeperpart of the annulus fibrosus or the nucleus pulposus in normal discs,nerves may reach these parts in degenerated discs through annular tears.

[0018] Silent disc herniations

[0019] As presented earlier, it is known that approximately one-third ofa normal adult population who never suffered from sciatica haveradiological visible disc herniations. Since the presence of a discherniation is so intimately linked to the symptom of sciatica this issurprising, and at present there is no valid explanation for thisphenomenon. However, “silent” in this regard only implies that the discherniations did not produce sciatica. One may assume though that theyproduce other symptoms.

[0020] Whiplash and whiplash associated disorders (WAD)

[0021] About 10% to 20% of the occupants of a stricken vehicle inrear-end car collisions suffer from whiplash injury. The injury may alsooccur as a result of other types of accidents, such as train accidents,and sudden retardations. This injury is defined as a non-contactacceleration-deceleration injury to the head-neck system. It is mostoften caused by a rear-end car collision and there is no direct impacton the neck.

[0022] Presenting symptoms usually include neckpain, headaches,disequilibrium, blurred vision, parenthesize, changes in cognition,fatigue, insomnia and hypersensitivity to light and sound. Dizzinessdescribed in a variety of terms such as imbalance, light-headedness andvertigo also occur frequently and these symptoms may be associated withlong-term disability.

[0023] Although neurologic and orthopedic examinations do not revealabnormalities in the majority of patients, the characteristics ofdizziness due to whiplash can be elucidated by means ofElectroNystagmoGraphic (ENG) evaluation. This examination is a methodthat is suitable for proving pathology in the oculo-vestibular system ofwhiplash-patients.

[0024] Until recently, the reason for the extent of injury was poorlyunderstood. In addition, due to the legal and insurance issues, theveracity of complaints of neck pain and other symptoms by people whosuffer from whiplash is commonly viewed as suspect.

[0025] Whiplash injuries can be quite complex and may include a varietyof related problems, such as joint dysfunction, and faulty movementpatterns, chronic pain and cognitive and higher center dysfunction.

[0026] When the cervical spine (neck) is subject to a whiplash injury,there is usually a combination of factors that contribute to the pain.These factors must be addressed individually, while maintaining a“holistic” view of the patient.

[0027] The most significant factors may include one or more of thefollowing: joint dysfunction, muscle dysfunction, and faulty movementpatterns.

[0028] Joint dysfunction

[0029] This occurs when one of the joints in the spine or limbs losesits normal joint play (resiliency and shock absorption). It is detectedthrough motion palpation, a procedure in which the doctor gently movesthe joint in different directions and assesses its joint play. When ajoint develops dysfunction, its normal range of movement may be affectedand it can become painful. In addition, joint dysfinction can lead to amuscle imbalance and muscle pain and a vicious cycle. The loss of jointplay can cause abnormal signals to the nervous system (there are anabundance of nerve receptors in the joint). The muscles related to thatjoint can subsequently become tense or, conversely, underactive. Theresulting muscle imbalance can place increased stress on the joint,aggravating the joint dysfunction that already exists.

[0030] Muscle dysfunction

[0031] When joint dysfunction develops, muscles are affected. Somemuscles respond by becoming tense and overactive, while others respondby becoming inhibited and underactive. In either case, these muscles candevelop trigger points. Trigger points are areas of congestion withinthe muscle where sensitizing compounds accumulate. These sensitizingcompounds can irritate the nerve endings within the muscle and producepain. This pain can occur in the muscle itself or can be referred pain(perceived in other areas of the body). Muscle related mechanisms mayalso give rise to abnormal signaling to the nervous system. This eventcan subsequently cause disruption of the ability of the nervous systemto properly regulate muscles in other parts of the body, leading to thedevelopment of faulty movement patterns.

[0032] Faulty movement patterns

[0033] It is thought that the intense barrage of pain signals from atraumatic injury to the cervical spine can change the way the nervoussystem controls the coordinated function of muscles. The disruption ofcoordinated, stable movement is known as faulty movement patterns.Faulty movement patterns cause increased strain in the muscles andjoints, leading to pain. They can involve the neck itself or can arisefrom dysfunction in other areas of the body such as the foot or pelvis.Instability is also considered part of faulty movement patterns. Thereare 2 types of instability that can occur in whiplash: passiveinstability-the ligaments of the neck are loosened, and dynamicinstability-the nervous system disruption causes a disturbance in thebody's natural muscular response to common, everyday forces. As a resultof instability, even mild, innocuous activities can become painful.

SUMMARY OF THE INVENTION

[0034] It has been found that the use of a TNF-alpha inhibitor, such asa substance selected from the group consisting of metalloproteinaseinhibitors excluding methylprednisolone, tetracyclines includingchemically modified tetracyclines, quinolones, corticosteroids,thalidomide, lazaroids, pentoxifylline, hydroxamic acid derivatives,carbocyclic acids, napthopyrans, soluble cytokine receptors, monoclonalantibodies towards TNF-alpha, amrinone, pimobendan, vesnarinone,phosphodiesterase inhibitors, lactoferrin and lactoferrin derivedanalogs, and melatonin are suitable for treatment of spinal disordersand nerve root injury caused by the liberation of TNF-alpha andcompounds triggered by the liberation of or presence of TNF-alpha byinhibiting spinal disk TNF-alpha.

[0035] These substances are thus suitable for treatment of nerve rootinjury, and for treatment of sciatica, low back pain (LBP), and whiplashassociated disorder (WAD).

[0036] TNF is one of many pro-inflammatory substances with similaraction, and it is considered as a “major player” in inflammatory events.However, TNF may also in part acts through other pro-inflammatorycytokines such as for instance IL-1, IL-6, FAS, and IFN-gamma.

[0037] The present invention is based on the finding that by combiningat least two different inhibitors of pro-inflammatory cytokines it ispossible to provide an even better treatment of the above-mentioneddiseases and conditions.

[0038] It is an object of the invention to provide novel and improvedmethods for inhibiting the action of cytokines for treating of nervedisorders in a subject comprising the step of administering to saidsubject a therapeutically effective dosage of at least two substancesselected from the group consisting of TNF inhibitors, IL-1 inhibitors,IL-6 inhibitors, IL-8 inhibitors, FAS inhibitors, FAS ligand inhibitors,and IFN-gamma inhibitors.

[0039] A preferred embodiment of the invention is a method for treatmentof a nerve disorder in a subject comprising administering to a subject atherapeutically effective dosage of a TNF inhibitor in combination witha second inhibitor selected from the group consisting of IL-1inhibitors, IL-6 inhibitors, IL-8 inhibitors, FAS inhibitors, FAS ligandinhibitors, and IFN-gamma inhibitors.

[0040] Another preferred embodiment of the invention is a method fortreatment of a nerve disorder in a subject comprising administering to asubject a therapeutically effective dosage of one TNF inhibitor, such asa specific TNF inhibitor, in combination with another TNF inhibitor,such as a non-specific TNF inhibitor.

[0041] Another preferred embodiment of the invention is a method fortreatment of a nerve disorder in a subject comprising administering to asubject a therapeutically effective dosage of a TNF inhibitor incombination with a IL-1 inhibitor.

[0042] It is also an object of the invention to provide a novelpharmaceutical composition for treating nerve disorders in a subjectcomprising a therapeutically effective dosage of at least two substancesselected from the group consisting of TNF inhibitors, IL-1 inhibitors,IL-6 inhibitors, IL-8 inhibitors, FAS inhibitors, FAS ligand inhibitors,and IFN-gamma inhibitors.

[0043] Nerve disorders treatable with the method and the pharmaceuticalcomposition according to the invention are nerve disorders due to areduced nerve reduction velocity, spinal disorders, nerve root injuries,nerve disorders caused by disk herniation, sciatica, cervicalrhizopathy, low back pain, whiplash associated disorder, nerve disordersinvolving pain, nucleus pulposus-induced nerve injuries, and spinal cordcompressions.

[0044] The subject which can be treated by these methods include anyvertebrate, preferably mammals, and of those, most preferably humans.

[0045] Although a break-through in the treatment of spinal painsyndromes was made in 1997 when the involvement of pro-inflammatorycytokines, in particular TNF, became evident, the current inventionoffers an even more efficient way to treat i.a. sciatica and low backpain by pharmacological means. Since TNF acts synergistically with otherpro-inflammatory cytokines inhibition of more cytokines than TNF is moreefficient in acquiring the desired clinical effect.

[0046] With the foregoing and other objects, advantages and features ofthe invention that will become hereinafter apparent, the nature of theinvention may be more clearly understood by reference to the followingdetailed description of the preferred embodiments of the invention andto the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

[0047] It has now surprisingly been shown possible to be able to treatnerve root injuries, or at least alleviate the symptoms of nerve rootinjuries by using a pharmaceutical composition comprising atherapeutically active amount of a TNF-alpha inhibitor. TNF-alphainhibitors, include but are not limited to, metalloproteinase (MMP)inhibitors (excluding methylprednisolone), tetracyclines, chemicallymodified tetracyclines, quinolones, corticosteroids, thalidomide,lazaroids, pentoxifylline, hydroxamic acid derivatives, napthopyrans,soluble cytokine receptors, monoclonal antibodies towards TNF-alpha,amrinone, pimobendan, vesnarinone, phosphodiesterase inhibitors,lactoferrin and lactoferrin derived analogous, and melatonin in the formof bases or addition salts together with a pharmaceutically acceptablecarrier.

[0048] By “therapeutically active amount” and “therapeutically effectivedosage” are intended to be an amount that will lead to a desiredtherapeutic effect, i.e., an amount that will lead to an improvement ofthe patient's condition. In one preferred example, an amount sufficientto ameliorate or treat a condition associated with a nerve disorder.

[0049] By “mammal” is meant to include but is not limited to primate,human, canine, porcine, equine, murine, feline, caprine, ovine, bovine,lupine, camelid, cervidae, rodent, avian and ichthyes. By animal ismeant to include any vertebrate animal wherein there is a potential fornerve root injury.

[0050] As used herein, the term “antibody” is meant to refer tocomplete, intact antibodies, and Fab fragments, scFv, and F(ab)₂fragments thereof. Complete, intact antibodies include monoclonalantibodies such as murine monoclonal antibodies (mAb), chimericantibodies, humanized antibodies and human. The production of antibodiesand the protein structures of complete, intact antibodies, Fabfragments, scFv fragments and F(ab)₂ fragments and the organization ofthe genetic sequences that encode such molecules, are well known and aredescribed, for example, in Harlow et al., ANTIBODIES: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988)and Harlow et al., USING ANTIBODIES: A LABORATORY MANUAL, Cold SpringHarbor Press, 1999, which are herein incorporated by reference in theirentirety.

[0051] By “epitope” is meant a region on an antigen molecule to which anantibody or an immunogenic fragment thereof binds specifically. Theepitope can be a three dimensional epitope formed from residues ondifferent regions of a protein antigen molecule, which, in a naivestate, are closely apposed due to protein folding. “Epitope” as usedherein can also mean an epitope created by a peptide or hapten portionof TNF-alpha and not a three dimensional epitope. Preferred epitopes arethose wherein when bound to an immunogen (antibody, antibody fragment,or immunogenic fusion protein) results in inhibited or blocked TNF-alphaactivity.

[0052] By “TNF-alpha blocking” is meant a compound or composition thatblocks, inhibits or prevents the activity of TNF or TNF-alpha.

[0053] Compounds that possess TNF-alpha inhibitory activity are forexample tetracyclines, (e.g., tetracycline, doxycycline, lymecycline,oxytetracycline, minocycline), and chemically modified tetracyclines(e.g., dedimethylamino-tetracycline), hydroxamic acid compounds,carbocyclic acids and derivatives, thalidomide, lazaroids,pentoxifylline, napthopyrans, soluble cytokine receptors, monoclonalantibodies towards INF-alpha, amrinone, pimobendan, vesnarinone,phosphodiesterase inhibitors, lactoferrin and lactoferrin derivedanalogs, melatonin, norfloxacine, ofloxacine, ciprofloxacine,gatifloxacine, pefloxacine, lomefloxacine, temafloxacine, TTP and p38kinase inhibitors. These compounds can be present as bases or in theform of addition salts, whichever possesses the best or preferredpharmaceutical effect, and best property to be brought into a suitablepharmaceutical composition. A more complete list is given below.

[0054] As stated above, there are several different types of cytokineblocking substances and pharmacological preparations that may be usedaccording to the invention, and those substances may be grouped indifferent subclasses:

[0055] Also contemplated are the pharmaceutically acceptable bases andsalts of the substances listed above.

[0056] Preferred groups of TNF-alpha blocking substances for useaccording to the present invention are soluble cytokine receptors,monoclonal antibodies, and tetracyclines or chemically modifiedtetracyclines.

[0057] Two preferred substances for use according to the presentinvention are the monoclonal antibodies, D2E7 and CDP-870.

[0058] D2E7 is a fully humanized monoclonal antibody directed againsthuman TNF-alpha, which has been developed by Knoll and CambridgeAntibody Technology. A transgenic recombinant version of this antibodyis under development by Genzyme Transgenic. The invention contemplatesany antibody that binds to the same epitope as D2E7 or that has the sameTNF-alpha inhibitory effect as D2E7. Preferably the antibody isprimatized®, humanized or human.

[0059] CDP-870 (or CDP 870) is a humanized antibody fragment with highaffinity to TNF-alpha. It has been developed by Celltech Group plc, andis co-developed with Pharmacia Corporation. The invention contemplatesany antibody, antibody fragment or immunogen that binds to the sameepitope as CDP-870 or that has the same TNF-alpha inhibitory activity asCDP-870. Preferably the antibody, antibody fragment or immunogen has thesame or similar TNF-alpha inhibitory activity. Preferably the antibody,antibody fragment or immunogen is primatized, humanized or human.

[0060] According to a preferred embodiment, one of the substances usedis a TNF inhibitor. According to a preferred variant of this embodimentthe TNF inhibitor is a monoclonal antibody directed against TNF, such asinfliximab, CDP-571, D2E7 or CDP-870. According to another preferredvariant of this embodiment the TNF inhibitor used is a soluble cytokineTNF receptor, such as etanercept. According to another preferred variantof this embodiment the TNF inhibitor used is a binuclear DNA threadingtransition metal complex with anti-cancer effect. According to anotherpreferred variant of this embodiment the TNF inhibitor used is alactoferrin derivable peptide. According to another preferred variant ofthis embodiment the TNF inhibitor used is an MMP inhibitor, such asdoxycycline. According to another preferred variant of this embodimentthe TNF inhibitor used is a p38 kinase inhibitor. According to yetanother preferred variant of this embodiment the substance used is TTP.

[0061] According to another preferred embodiment, one of the substancesis a specific TNF inhibitor, such as infliximab, CDP-571, D2E7 orCDP-870, which is use in combination with a non-specific TNF inhibitor,such as doxycycline.

[0062] Doxycycline inhibits the action of TNF in a non-specific manner.TNF and other similar bioactive substances are first produced in aninactive form and transported to the cell membrane. Upon activation, theactive part of the pro-TNF is cleaved and released. This process iscalled shedding and may be initiated by one or more enzymes. Theseenzymes have in common that they are metalloproteinases, i.e. dependentof a metal-ion for their function. Doxycycline and other tetracyclinesare known to bind to metal-ions and will thereby inhibit the action ofmetalloproteinases and subsequently the release of TNF and otherpro-inflammatory cytokines in a non-specific manner. A monoclonalanti-TNF antibody, on the other hand, will bind directly to TNF andthereby inhibit TNF in a more specific way than doxycycline. Theinhibition may thus be assumed to be more efficient but will berestricted to TNF. However, in the work leading to the presentinvention, it was found that anti-TNF treatment was more efficient thandoxycycline treatment. However, the most efficient way to inhibit thenucleus pulpous induced reduction in nerve root conduction velocity wasfound to be to combine a TNF-inhibitor and an IL-1 inhibitor. TNF isknown to be orchestrating much of the inflammatory events. Except forhaving direct effects on target-receptors, it may also act through othercytokines by stimulation of their release. Although one may assume thatspecific inhibition may block both the direct effects of TNF and itsstimulatory effects on other cytokines, the work leading to the presentinvention showed that it is possible to further inhibit the action ofTNF by inhibiting cytokines that are produced and released by TNFstimulation. In addition to inhibiting the stimulatory effects of TNF,one thereby also inhibits the effects of these synergistic cytokinesdownstream. Since these synergistic cytokines may also induce releaseand production of TNF, the inhibition of these cytokines also reducesthe level of TNF. According to the present invention it is shown thatthe use of also one or more inhibitors of other cytokines is moreefficient in blocking the nucleus pulposus induced effects on adjacentnerve roots.

[0063] The combination of inhibitors of one specific cytokine withdifferent mechanisms is also shown to be more efficient than the use ofa single inhibitor. For instance, TNF may be inhibited at thesynthesis-level (e.g., by pentoxifylline), at translation by anti-sense(e.g., by ISIS-104838), at shedding (e.g., by doxycycline), and later byantibodies (e.g., by infliximab or CDP-870) or soluble receptors (e.g.,by etanercept or lenercept). Thus, there are at least five mechanismsuseful for inhibiting TNF. The combination of two or more drugs that actthrough different mechanisms therefore induces a more efficientinhibition of that certain cytokine than the use of one single drug. Aspecial benefit is achieved if a specific inhibitor or a key substance,for instance a monoclonal antibody against TNF, and a non-specificinhibitor that also blocks other cytokines, for instance doxycycline.This combination achieves inhibition of TNF at two levels (shedding andbinding of active TNF) as well as inhibition of other synergisticcytokines.

[0064] According to another preferred embodiment one of the substancesused is an IL-1 inhibitor.

[0065] According to an especially preferred embodiment one of thesubstances used is a TNF inhibitor and one is an IL-1 inhibitor.

[0066] Said at least two substances are preferably administeredsimultaneously, but they may also be administered separately.

[0067] The substances according to the invention may also beadministered in combination with other drugs or compounds, provided thatthese other drugs or compounds do not eliminate the desired effectsaccording to the present invention, i.e., the effect on TNF-alpha.

[0068] The invention further relates to a method for inhibiting thesymptoms of nerve root injury.

[0069] The effects of doxycycline, soluble cytokine-receptors, andmonoclonal cytokine-antibodies have been studied and representativemethods used and results obtained are disclosed below. Although thepresent invention has been described in detail with reference toexamples herein, it is understood that various modifications can be madewithout departing from the spirit of the invention, and would be readilyknown to the skilled artisan.

[0070] The compounds of the invention can be administered in a varietyof dosage forms, e.g., orally (per os), in the form of tablets,capsules, sugar or film coated tablets, liquid solutions; rectally, inthe form of suppositories; parenterally, e.g., intramuscularly (i.m.),subcutaneous (s.c.), intracerebroventricular (i.c.v.), intrathecal(i.t.), epidurally, transepidermally or by intravenous (i.v.) injectionor infusion; by inhalation; or intranasally.

[0071] The therapeutic regimen for the different clinical syndromes maybe adapted to the disease or condition, medical history of the subjectas would be know to the skilled artisan or clinician. Factors to beconsidered but not limiting to the route of administration, the form inwhich the compound is administered, the age, weight, sex, and conditionof the subject involved.

[0072] For example, the oral route is employed, in general, for allconditions, requiring such compounds. In emergency cases, preference issometimes given to intravenous injection. For these purposes, thecompounds of the invention can be administered, for example, orally atdoses ranging from about 20 to about 1500 mg/day. Of course, thesedosage regimens may be adjusted to provide the optimal therapeuticresponse depending on the subject's condition.

[0073] The nature of the pharmaceutical composition containing thecompounds of the invention in association with pharmaceuticallyacceptable carriers or diluents will, of course, depend upon the desiredroute of administration. The composition may be formulated in theconventional manner with the usual ingredients. For example, thecompounds of the invention may be administered in the form of aqueous oroily solutions or suspensions, tablets, pills, gelatin capsules (hard orsoft ones), syrups, drops or suppositories.

[0074] For oral administration, the pharmaceutical compositionscontaining the compounds of the invention are preferably tablets, pillsor gelatine capsules, which contain the active substance or substancestogether with diluents, such as lactose, dextrose, sucrose, mannitol,sorbitol, cellulose; lubricants, e.g., silica, talc, stearic acid,magnesium or calcium stearate, and/or polyethylene glycols; or they mayalso contain binders, such as starches, gelatine, methyl cellulose,carboxymethylcellulose, gum arabic, tragacanth, polyvinylpyrrolidone;disaggregating agents such as starches, alginic acid, alginates, sodiumstarch glycolate, microcrystalline cellulose; effervescing agents, sucha carbonates and acids; dyestuffs; sweeteners; wetting agents, such aslecithin, polysorbates, laurylsulphates; and in general non-toxic andpharmaceutically inert substances used in the formulation ofpharmaceutical compositions. Said pharmaceutical compositions may bemanufactured in known manners, e.g., by means of mixing, granulating,tableting, sugar-coating or film-coating processes. Film providingcompounds can be selected to provide release in the right place or atthe appropriate time in the intestinal tract with regard to absorptionand maximum effect. Thus pH-dependent film formers can be used to allowabsorption in the intestines as such, whereby different phthalates arenormally used or acrylic acid/methacrylic acid derivatives and polymers.

[0075] The liquid dispersions for oral administration may be, e.g.,syrups, emulsions, and suspensions.

[0076] The syrups may contain as carrier, e.g., saccharose, orsaccharose with glycerine and/or mannitol and/or sorbitol.

[0077] Suspensions and emulsions may contain as Garner, e.g., a naturalgum, such as gum arabic, xanthan gum, agar, sodium alginate, pectin,methyl cellulose, carboxymethylcellulose, polyvinyl alcohol.

[0078] The suspension or solutions for intramuscular injections maycontain together with the active compound, a pharmaceutically acceptablecarrier, such as e.g., sterile water, olive oil (or other vegetable ornut derived oil), ethyl oleate, glycols”, e.g., propylene glycol, and ifso desired, a suitable amount of lidocaine hydrochloride. Adjuvants fortriggering the injection effect can be added as well.

[0079] The solutions for intravenous injection or infusion may containas carrier, e.g., sterile water, or preferably, a sterile isotonicsaline solution, as well as adjuvants used in the field of injection ofactive compounds. Such solutions would also be suitable for i.m. andi.c.v. injection.

[0080] The suppositories may contain together with the active compounds,a pharmaceutically acceptable carrier, e.g., cocoa-butter polyethyleneglycol, a polyethylene sorbitan fatty acid ester surfactant or lecithin.

[0081] Examples of suitable doses of the active agents contemplated fordifferent administration routes are given below. Per os 10-300 mg i.m.25-100 mg i.v. 2.5-25 mg i.t. 0.1-25 mg (daily—every 3^(rd) month)inhalation 0.2-40 mg transepidermally 10-100 mg intranasally 0.1-10 mgs.c. 5-10 mg i.c.v. 0.1-25 mg (daily—every 3^(rd) month) epidurally1-100 mg

[0082] These ranges are approximate (e.g., about 1 to about 100) and mayvary depending on the specific agent being administered and the natureof the disorder in the subject. Thus, it is further contemplated thatany dosage in between for the cited ranges may also be used.

[0083] Examples of suitable doses for different TNF-alpha inhibitors aregiven in the table below. More Most Preferred preferred preferred dosagedosage dosage TNF-alpha blocking substance and administration routeLenercept i.v.  5-200  10-100 30-80 (all given in mg for administrationonce every 4th week) TBP-1 i.v.  5-200  10-100 30-80 (all given in mgfor administration once every 4th week) CDP-571 (HUMICADE ®) i.v.  1-100 5-10  5-10 (all given in mg/kg body weight for administration as asingle dose) D2E7 i.v. 0.1-50  0.5-10   1-10 s.c. 0.1-50  0.5-10   1-10(all given in mg/kg body weight for administration as a single dose)Iloprost i.v.  0.1-2000    1-1500  100-1000 (all given in μg/kg bodyweight/day) intranasally  50-250 100-150 100-150 (all given in μg/day)Thalidomide  100-1200  300-1000 500-800 (all given in μg/day) CC-1088Per os  50-1200 200-800 400-600 (all given in mg/day) CDP-870 i.v.  1-50 2-10 3-8 (all given in mg/kg body weight for administration once every4th week) HP-228 i.v.  5-100 10-50 20-40 (all given in μg/kg bodyweight) ISIS-10483 Per os  1-100 10-50 20-50 S.c.  1-100 10-50 20-50i.v.  1-100 10-50 20-50 (all given in mg) ARIFLO ® (SB 207499 Per os 10-100 30-60 30-45 (all given in mg/day) KB-R7785 s.c. 100-500 100-300150-250 (all given in mg/kg body weight/day) CDC-501 Per os  50-1200200-800 400-600 (all given in mg/day) CDC-801 (ROQUININEX ®) Per os 50-1200 200-800 400-600 (all given in mg/day) Prinomastat, Batimastat,and Marimastat Per os 1-250 mg 5-100 mg 10-50 mg (all given in mgtwice/day) Linomide Per os 0.1-25   5-20 110-15  (all given in mg/kgbody weight/day) IL-1 blocking substance and administration routeAnakinra (KINERET ®) s.c.  10-200  50-150 100 (all given in mg/day)

[0084] Incorporation by Reference and Examples

[0085] Although the present invention has been described in detail withreference to examples below, it is understood that various modificationscan be made without departing from the spirit of the invention, andwould be readily known to the skilled artisan. All cited patents andpublications referred to in this application are herein incorporated byreference in their entirety for all purposes.

[0086] The application incorporates herein by reference in theirentirety International Application No. PCT/SE99/01670 and SwedishApplication Nos. 9803276-6 and 9803710-4 for all purposes.

EXAMPLES Example 1

[0087] Study design

[0088] The effects of nucleus pulposus and various treatments to blockTNF-activity were evaluated in an experimental set-up usingimmunohistochemistry and nerve conduction velocity recordings.

[0089] Summary of background data

[0090] A meta-analysis of observed effects induced by nucleus pulposusreveals that these effects might relate to one specific cytokine, TumorNecrosis Factor alpha (TNF-alpha).

[0091] Objectives

[0092] To assess the presence of TNF-alpha in pig nucleus pulposus cellsand to see if blockage of TNF-alpha also blocks the nucleuspulposus-induced reduction of nerve root conduction velocity.

[0093] Methods

[0094] Series-1: Cultured nucleus pulposus-cells wereimmunohistologically stained with a monoclonal antibody for TNF-alpha.

[0095] Series-2: Nucleus pulposus was harvested from lumbar discs andapplied to the sacrococcygeal cauda equina in 13 pigs autologously. Fourpigs received 100 mg of doxycycline intravenously, 8 pigs had a blockingmonoclonal antibody to TNF-alpha applied locally in the nucleuspulposus, and 4 pigs remained non-treated (controls). Three days afterthe application the nerve root conduction velocity was determined overthe application zone by local electrical stimulation.

[0096] Series-3: Thirteen pigs had autologous nucleus pulposus placedonto their sacrococcygeal cauda equina similar to series-2. Five pigs(body weight 25 kg) received REMICADE® (infliximab) 100 mg i.v.preoperatively, and 8 pigs received ENBREL® (etanercept) 12.5 mg s.c.preoperatively and additionally 12.5 mg s.c. three days after theoperation. Seven days after the nucleus pulposus-application the nerveroot conduction velocity was determined over the application zone bylocal electrical stimulation according to series-2.

[0097] Results

[0098] Series-1: TNF-alpha was found to be present in the nucleuspulposus-cells.

[0099] Series-2: The selective antibody to TNF-alpha limited thereduction of nerve conduction velocity. However, treatment withdoxycycline significantly blocked the nucleus pulposus-induced reductionof conduction velocity.

[0100] Series-3: Both drugs (infliximab, and etanercept) blocked thenucleus pulposus induced nerve injury efficiently. Normal average nerveconduction velocities were found after 15 treatment with both of thesetwo drugs.

[0101] Conclusion

[0102] For the first time a specific substance, Tumor NecrosisFactor-alpha (TNFα), has been linked to the nucleus pulposus-inducedeffects of nerve roots after local application. Although the effects ofthis substance may be synergistic with other similar substances, thedata of the present study may be of significant importance for thecontinued understanding of nucleus pulposus' biologic activity, andmight also be of potential use for future treatment strategies ofsciatica and other nerve root injury conditions or related conditions.

[0103] After previously being considered as just a biologically inactivetissue component compressing the spinal nerve root at disc herniation,the nucleus pulposus has recently been found to be highly active,inducing both structural and functional changes in adjacent nerve rootswhen applied epidurally (Kayama S, Konno S, Olmarker K, Yabuki S,Kikuchi S, Incision of the anulus fibrosis induces nerve rootmorphologic, vascular, and functional changes. An experimental study,Spine 1996; 21: 2539-43; Olmarker K, Brisby H, Yabuki S, Nordborg C,Rydevik B, The effects of normal, frozen, and hyaluronidase-digestednucleus pulposus on nerve root structure and function, Spine 1997; 22:4715; discussion 476; Olmarker K, Byrod G, Comefjord M, Nordborg C,Rydevik B, Effects of methylprednisolone on nucleus pulposus-inducednerve root injury, Spine 1994; 19: 1803-8; Olmarker K, Nordborg C,Larsson K, Rydevik B, Ultrastructural changes in spinal nerve rootsinduced by autologous nucleus pulposus, Spine 1996; 21: 411-4; OlmarkerK, Rydevik B, Nordborg C, Autologous nucleus pulposus inducesneurophysiologic and histologic changes in porcine cauda equina nerveroots, Spine 1993; 18: 1425-32). It has thereby been established thatautologous nucleus pulposus may induce axonal changes and acharacteristic myelin injury (Kayama S, Konno S, Olmarker K, Yabuki S,Kikuchi S, Incision of the anulus fibrosis induces nerve rootmorphologic, vascular, and functional changes. An experimental study,Spine 1996; 21: 2539-43; Olmarker K, Byrod G, Comefjord M, Nordborg C,Rydevik B, Effects of methylprednisolone on nucleus pulposus-inducednerve root injury, Spine 1994; 19: 1803-8; Olmarker K, Nordborg C,Larsson K, Rydevik B, Ultrastructural changes in spinal nerve rootsinduced by autologous nucleus pulposus, Spine 1996; 21: 411-4; OlmarkerK, Rydevik B, Nordborg C, Autologous nucleus pulposus inducesneurophysiologic and histologic changes in porcine cauda equina nerveroots, Spine 1993; 18: 1425-32.), increased vascular permeability (ByrodG, Otani K, Brisby H, Rydevik B, Olmarker K, Methylprednisolone reducesthe early vascular permeability increase in spinal nerve roots inducedby epidural nucleus pulposus application, J Orthop Res 1987; 18: 6:983-7), infra vascular coagulation (Kayama S, Konno S, Olmarker K,Yabuki S, Kikuchi S, Incision of the anulus fibrosis induces nerve rootmorphologic, vascular, and functional changes. An experimental study,Spine 1996; 21: 2539-43; Olmarker K, Blomquist J, Stromberg J, Nanmnark,U, Thomsen P, Rydevik B, Inflammatogenic properties of nucleus pulposus,Spine 1995; 20: 665-9.), and that membrane-bound structure or substancesof the nucleus pulposus-cells are responsible for these effects (KayamaS, Konno S, Olmarker K, Yabuki S, Kikuchi S, Incision of the anulusfibrosis induces nerve root morphologic, vascular, and functionalchanges. An experimental study, Spine 1996; 21: 2539-43; Olmarker K,Brisby H, Yabuki S, Nordborg C, Rydevik B, The effects of normal,frozen, and hyaluronidase-digested nucleus pulposus on nerve rootstructure and function, Spine 1997; 22: 4715; discussion 476.). Theeffects have also been found to be efficiently blocked bymethylprednisolone and cyclosporin A (Arai I, Konno S, Otani K, KikuchiS, Olmarker K, Cyclosporin A blocks the toxic effects of nucleuspulposus on spinal nerve roots, Submitted; Olmarker K, Byrod G,Comefjord M, Nordborg C, Rydevik B, Effects of methylprednisolone onnucleus pulposus-induced nerve root injury, Spine 1994; 19: 1803-8.).When critically looking at these data, one realizes that there is atleast one cytokine that relates to all of these effects, TNF-alpha.

[0104] To assess if TNF-alpha may be involved in the nucleus pulposusinduced nerve root injury, the presence of TNF-alpha in nucleuspulposus-cells was assessed and was studied if the nucleuspulposus-induced effects could be blocked by doxycycline, a solubleTNF-receptor, and a selective monoclonal TNF-alpha antibody, the latteradministered both locally in the nucleus pulposus and systemically.

Example 2

[0105] Material and Methods

[0106] Series-1, Presence of TNF-alpha in pig nucleus pulposus-cells:

[0107] Nucleus pulposus (NP) from a total of 13 lumbar and thoracicdiscs were obtained from 10 pigs, which were used for other purposes. NPwas washed once in Ham's F12 medium (Gibco BRL, Paisley, Scotland) andthen centrifuged and suspended in 5 ml of collagenase solution in Ham'sF12 medium (0.8 mg/ml, Sigma Chemical Co., St Louis, Mo., USA) for 40minutes, at 37° C. in 25 cm tissue culture flasks. The separated NP-cellpellets were suspended in DMEM/F12 1:1 medium (Gibco BRL, Paisley,Scotland) supplemented with 1% L-glutamine 200 mM (Gibco BRL, Paisley,Scotland), 50 mg/ml gentamycine sulphate (Gibco BRL, Paisley, Scotland)and 10% fetal calf serum (FCS), (Gibco BRL, Paisley, Scotland). Thecells were cultured at 37° C. and 5% CO₂ in air for 3-4 weeks and thencultured directly on tissue culture treated glass slides (BectonDickinson & Co Labware, Franklin Lakes, N.J., USA). After 5 days on theglass slides, the cells were fixed in situ by exposing the slides toacetone for 10 minutes. After blocking irrelevant antigens byapplication of 3% H₂O₂ (Sigma Chemical Co., St Louis, Mo., USA) for 30minutes and Horse Serum (ImmunoPure ABC, peroxidase mouse IgG stainingkit nr.32028, Pierce, Rockford, Ill.) for 20 minutes, the primaryantibody (Anti-pig TNF-alpha monoclonal purified antibody, Endogen,Cambridge, Mass., USA, Ordering Code MP-390) was applied over night at+40° C., diluted at 1:10, 1:20 and 1:40 dilutions. For control, BSA(bovine serum albumin, Intergen Co, New York, USA) suspended in PBS(phosphate buffered saline, Merck, Darmstadt, Germany) was applied inthe same fashion. The next day the cells were washed with 1% BSA in PBSand the secondary antibody (ImmunoPure ABC, peroxidase mouse IgGstaining kit Cat. Cat. #32028, Pierce, Rockford, Ill.) was applied for30 minutes. To enhance this reaction, the cells were exposed toAvidin-Biotin complex for an additional 30 minutes (ImmunoPure ABC,peroxidase mouse IgG staining kit Cat. #32028, Pierce, Rockford, Ill.).The cells were then exposed to 20 mg of DAB (3,3-diaminobenzidinetetrahydrochloride No. D-5905, Sigma Chemical Co., St Louis, Mo., USA)and 0.033 ml of 3% H₂O₂ in 10 ml of saline for 10 minutes. The cellswere washed in PBS, dehydrated in a series of ethanol, mounted andexamined by light microscopy by an unbiased observer for the presence ofa brown coloration indicating the presence of TNF-alpha.

[0108] Series-2, Neurophysiologic evaluation:

[0109] Thirteen pigs (body weight 25-30 kg) received an intramuscularinjection of 20 mg/kg body weight of KETALAR® (ketamine, 50 mg/ml,Parke-Davis, Moms Plains, N.J.) and an intravenous injection of 4 mg/kgbody weight of HYPNODIL® (methomidate chloride, 50 mg/ml, AB Leo,Helsingborg, Sweden) and 0.1 mg/kg body weight of STRESNIL® (azaperon, 2mg/ml, Janssen Pharmaceutica, Beerse, Belgium). Anesthesia wasmaintained by additional intravenous injections of 2 mg/kg body weightof HYPNODIL® and 0.05 mg/kg body weight of STRESNIL®. The pigs alsoreceived an intravenous injection of 0.1 mg/kg of STESOLID NOVUM®(Diazepam, Dumex, Helsingborg, Sweden) after surgery.

[0110] Nucleus pulposus was harvested from the 5^(th) lumbar discthrough a retro peritoneal approach (Olmarker K, Rydevik B, Nordborg C,Autologous nucleus pulposus induces neurophysiologic and histologicchanges in porcine cauda equina nerve roots, Spine 1993; 18: 1425-32.).Approximately 40 mg of the nucleus pulposus was applied to thesacrococcygeal cauda equina through a midline incision and laminectomyof the first coccygeal vertebra. Four pigs did not receive any treatment(no treatment). Four other pigs received an intravenous infusion of 100mg of doxycycline (Vibramycino, Pfizer Inc., New York, USA) in 100 ml ofsaline over 1 hour. In 5 pigs, the nucleus pulposus was mixed with 100μl of a 1.11 mg/mL suspension of the anti-TNF- alpha antibody used inseries 1, before application.

[0111] Three days after the application, the pigs were re-anesthetizedby an intramuscular injection of 20 mg/kg body weight of KETALAR® and anintravenous injection of 35 mg/kg body weight 25 of PENTOTHAL®(Thiopental sodium, Abbott lab, Chicago, Ill.). The pigs were ventilatedon a respirator. Anesthesia was maintained by an intravenous bolusinjection of 100 mg/kg body weight of Chloralose((a)-D(+)-gluco-chloralose, Merck, Darmstadt, Germany) and by acontinuous supply of 30 mg/kg/hour of Chloralose. A laminectomy from the4^(th) sacral to the 3^(rd) coccygeal vertebra was performed. The nerveroots were covered with SPONGOSTANE® (Ferrosan, Denmark). Local tissuetemperature was continuously monitored and maintained at 37.5-38.0° C.by means of a heating lamp.

[0112] The cauda equina was stimulated by two E2 subdermal platinumneedle electrodes (Grass Instrument Co., Quincy, Mass.) which wereconnected to a Grass SD9 stimulator (Grass Instrument Co., Quincy,Mass.) and gently placed intermittently on the cauda equina first 10 mmcranial and then 10 mm caudal to the exposed area. To ensure that onlyimpulses from exposed nerve fibers were registered, the nerve root thatexited from the spinal canal between the two stimulation sites were cut.An electromyogram (EMG) was registered by two subdermal platinum needleelectrodes which were placed into the paraspinal muscles in the tailapproximately 10 mm apart. This procedure is reproducible and representsa functional measurement of the motor nerve fibers of the cauda equinanerve roots. The EMG was visualized using a Macintosh IIci computerprovided with Superscope software and MacAdios II AID converter (GWInstruments, Sommerville, Mass.) together with a Grass P18 preamplifier(Grass Instrument Co., Quincy, Mass.). The separation distance betweenthe first peaks of the EMG from the two recordings was determined, andthe separation distance between the two stimulation sites on the caudaequina was measured with calipers. The nerve conduction velocity betweenthe two stimulation sites could thus be calculated from these twomeasurements.

[0113] The person performing the neurophysiologic analyses was unawareof the experimental protocol for the individual animal. After finishingthe complete study, the data were arranged in the three experimentalgroups and statistical differences between the groups were assessed byStudent's t-test. The experimental protocol for this experiment wasapproved by the local animal research ethics committee.

[0114] Series-3:

[0115] Thirteen pigs had autologous nucleus pulposus placed onto theirsacrococcygeal cauda equina similar to series-2. Five pigs (bodyweight25 kg) received the human/murine monoclonal antibody, REMICADE®(infliximab, Immunex Corporation, Seattle, Wash. 98101, USA) 100 mg i.v.preoperatively, and 8 pigs received ENBREL® (etanercept, Centocor B.V.,Leiden, the Netherlands) 12.5 mg s.c. preoperatively and additionally12.5 mg s.c. three days after the operation. Seven days after thenucleus pulposus-application the nerve root conduction velocity wasdetermined over the application zone by local electrical stimulationaccording to series-2. To blind the study, the neurophysiologicalevaluation was conducted in parallel to another study and the personperforming the analyses did not know from which study and what treatmenteach specific animal was subjected to. No non-treated animals wereincluded in the series-3 due to the pre-existing knowledge of nerveconduction velocity after seven days of either nucleus pulposus or fat(control) application. The statistical difference between the groups,infliximab, and etanercept, nucleus pulposus without treatment (positivecontrol from previous data) and application of retroperitoneal fat(negative control from previous data) was assessed by using ANOVA andFisher's PLSD at 5%.

[0116] RESULTS

[0117] Series-1, Presence of TNF-alpha in pig nucleus pulposus-cells:

[0118] Examples of the light microscopic appearance of the stained glassslides. In the sections using BSA in PBS as “primary antibody”(control), no staining was observed, ensuring that there was no labelingand visualization of irrelevant antigens. When the anti-TNF-alphaantibody was applied at 1:40 dilution there was only weak staining.However, the staining increased with diminishing dilutions of theantibody. The staining was seen in the soma of the cells, and it was notpossible to differentiate whether TNF-alpha was located in thecytoplasm, on the cell surface bound to the cell-membrane, or both.

[0119] Series-2 Neurophysiologic evaluation:

[0120] Application of non-modified nucleus pulposus and without anytreatment induced a reduction in nerve conduction velocity similar toprevious studies (Table 1). In contrast, treatment with doxycyclinecompletely blocked this reduction (p<0.01 Student's t-test). Localapplication of anti-TNF-alpha-antibody also induced a partial block ofthis reduction, although not as complete as doxycycline and was notstatistically significant as compared to the no treatment-series.

[0121] Series-3:

[0122] Treatment with both drugs seemed to prevent the nucleuspulposus-induced reduction of nerve root conduction velocities, sincethe average nerve conduction velocity for both these treatment groupswere close to the average conduction of the fat-application series, asseen in a previous study (Table 2). The average nerve conductionvelocity in pigs treated with ENBREL® was statistically different fromthe average nerve conduction velocity in the series with pigs with notreatment. The average new conduction velocity in the group treated withREMICADE® was also statistically significantly different from theaverage nerve conduction velocity in the group with no treatment. TABLE1 Series 2 Treatment n NCV (m/s ± SD) Local anti-TNF-alpha 5 64 ± 28Doxycycline 4 76 ± 9  No treatment 4 46 + 12

[0123] TABLE 2 Series 3 Treatment n NCV (m/s ± SD) Fat* 5 76 ± 11ENBREL ® 8 78 ± 14 REMICADE ® 5 79 ± 15 No treatment* 5 45 ± 19

[0124] Discussion

[0125] The data of the present study demonstrated that TNF-alpha may befound in nucleus pulposus-cells of the pig. If TNF-alpha was blocked bya locally applied selective monoclonal antibody, the nucleuspulposus-induced reduction of nerve root conduction velocity waspartially blocked, although not statistically significant as compared tothe series with non-treated animals. However, if animals were treatedsystemically with doxycycline, infliximab, and etanercept to inhibitTNF-alpha, the reduction of nerve conduction velocity was significantlyprevented.

[0126] In recent years, it has been verified that local application ofautologous nucleus pulposus may injure the adjacent nerve roots. Thus,it has become evident that the nerve root injury seen as disc herniationmay not be solely based on mechanical deformation of the nerve root, butmay also be induced by unknown “biochemical effects” related to theepidural presence of herniated nucleus pulposus. Although this newresearch field has generated many experimental studies, the mechanismsand substances involved are not fully known. It has been seen that localapplication of autologous nucleus pulposus may induce axonal injury(Kayama S, Konno S, Olmarker K, Yabuki S, Kikuchi S, Incision of theanulus fibrosis induces nerve root morphologic, vascular, and functionalchanges. An experimental study, Spine 1996; 21: 2539-43; Olmarker K,Brisby H, Yabuki S, Nordborg C, Rydevik B, The effects of normal,frozen, and hyaluronidase-digested nucleus pulposus on nerve rootstructure and function, Spine 1997; 22: 4715; discussion 476; OlmarkerK, Byrod G, Comefjord M, Nordborg C, Rydevik B, Effects ofmethylprednisolone on nucleus pulposus-induced nerve root injury, Spine1994; 19: 1803-8; Olmarker K, Myers R R, Pathogenesis of sciatic pain:Role of herniated nucleus pulposus and deformation of spinal nerve rootand DRG, Pain, 1998, 78: 9-105; Olmarker K, Nordborg C, Larsson K,Rydevik B, Ultrastructural changes in spinal nerve roots induced byautologous nucleus pulposus, Spine 1996; 21: 411-4; Olmarker K, RydevikB, Nordborg C, Autologous nucleus pulposus induces neurophysiologic andhistologic changes in porcine cauda equina nerve roots, Spine 1993; 18:1425-32), a characteristic injury of the myelin sheath (Kayama S, KonnoS, Olmarker K, Yabuki S, Kikuchi S, Incision of the anulus fibrosisinduces nerve root morphologic, vascular, and functional changes. Anexperimental study, Spine 1996; 21: 2539-43; Olmarker K, Byrod G,Comefjord M, Nordborg C, Rydevik B, Effects of methylprednisolone onnucleus pulposus-induced nerve root injury, Spine 1994; 19: 1803-8;Olmarker K, Myers R R, Pathogenesis of sciatic pain: Role of herniatednucleus pulposus and deformation of spinal nerve root and DRG, Pain,1998, 78: 9-105; Olmarker K, Nordborg C, Larsson K, Rydevik B,Ultrastructural changes in spinal nerve roots induced by autologousnucleus pulposus, Spine 1996; 21: 411-4; Olmarker K, Rydevik B, NordborgC, Autologous nucleus pulposus induces neurophysiologic and histologicchanges in porcine cauda equina nerve roots, Spine 1993; 18: 1425-32), alocal increase of vascular permeability (Byrod G, Otani K, Brisby H,Rydevik B, Olmarker K, Methylprednisolone reduces the early vascularpermeability increase in spinal nerve roots induced by epidural nucleuspulposus application, J. Orthop. Res. 1987; 186: 983-7; Olmarker K,Blomquist J, Stromberg J, Nanmnark, U, Thomsen P, Rydevik B,Inflammatogenic properties of nucleus pulposus, Spine 1995; 20: 665-9)infra vascular coagulations, reduction of infra neural blood flow (OtaniK, Arai I, Mao G P, Konno S, Olmarker K, Kikuchi S, Nucleuspulposus-induced nerve root injury. The relationship between blood flowand nerve conduction velocity, Neurosurgery 1999; 45: 619-20), andleukotaxis (Olmarker K, Blomquist J, Stromberg J, Nanmnark, U, ThomsenP, Rydevik B, Inflammatogenic properties of nucleus pulposus, Spine1995; 20: 665-9). It has been seen that the nucleus pulposus-relatedeffects may be blocked efficiently by methylprednisolone (Olmarker K,Byrod G, Comefjord M, Nordborg C, Rydevik B, Effects ofmethylprednisolone on nucleus pulposus-induced nerve root injury, Spine1994; 19: 1803-8) and cyclosporin A (Arai I, Konno S, Otani K, KikuchiS, Olmarker K, Cyclosporin A blocks the toxic effects of nucleuspulposus on spinal nerve roots, Submitted, and slightly less efficientlyby indomethacin (Arai I, Mao G P, Otani K, Komo S, Kikuchi S, OlmarkerK, Indomethacin blocks nucleus pulposus related effects in adjacentnerve roots, Accepted for publication in Eura Spine J.), and lidocaine(Yabuki S, Kawaguchi Y, Olmarker K, Rydevik B, Effects of lidocaine onnucleus pulposus-induced nerve root injury, Spine, 1998; 23: 29:2383-89). Further, it has been understood that the effects are mediatedby the nucleus pulposus-cells (37), particularly by substances orstructures bound to the cell-membranes (Kayama S, Olmarker K, Larsson K,Sjören-Jansson E, Lindahl A, Rydevik D, Cultured, autologous nucleuspulposus cells induce structural and functional changes in spinal nerveroots, Spine, 1998; 23: 2155-8). When critically considering these data,it becomes evident that at least one specific cytokine could be relatedto these observed effects, Tumor Necrosis Factor-alpha (TNF-alpha).TNF-alpha may induce nerve injury (Liberski P P, Yanagihara R, NerurkarV, Gajdusek D C, Further ultrastructural studies of lesions induced inthe optic nerve by tumor necrosis factor alpha (TNF-alpha): a comparisonwith experimental Creutzfeldt-Jakob disease, Acta Neurobiol En (Warsz)1994; 54: 209-18; Madigan M C, Sadun A A, Rao N S, Dugel P U, Tenhula WN, Gill P S, Tumor necrosis factor-alpha (TNF-alpha)-induced opticneuropathy in rabbits, Neurol Res 1996; 18: 176-84; Petrovich M S, Hsu HY, Gu X, Dugal P, Heller K B, Sadun A A, Pentoxifylline suppression ofTNF-alpha mediated axonal degeneration in the rabbit optic nerve, NeurolRes 1997; 19: 551-4; Said G, Hontebeyrie-Joskowicz M, Nerve lesionsinduced by macrophage activation, Res Immunol 1992; 143: 589-99; WagnerR, Myers R R, Endoneurial injection of TNF-alpha produces neuropathicpain behaviours, Neuroreport 1996; 7: 2897-901), mainly seen as acharacteristic myelin injury that closely resembles the nucleuspulposus-induced myelin-injury (Liberski P P, Yanagihara R, Nerurkar V,Gajdusek D C, Further ultrastructural studies of lesions induced in theoptic nerve by tumor necrosis factor alpha (TNF-alpha): a comparisonwith experimental Creutzfeldt-Jakob disease, Acta Neurobiol En (Warsz)1994; 54: 209-18; Redford E J, Hall S M, Smith K J, Vascular changes anddemyelination induced by the intraneural injection of tumour necrosisfactor, Brain 1995; 118: 869-78; Sehnaj K W, Raine C S, Tumor necrosisfactor mediates myelin and oligodendrocytc damage in vitro, Ann Neurol1988; 23: 339-46; Sharief M K, Ingram D A, Swash M, Circulating tumornecrosis factor-alpha correlates with electrodiagnostic abnormalities inGuillain-Barre syndrome, Ann Neurol 1997; 42: 6873; Tsukamoto T,Ishikawa M, Yamamoto T, Suppressive effects of TNF-alpha on myclinformation in vitro, Acta Neurol Scand 1995; 91: 71-5; Villarroya H,Violleau K, Ben Younes-Chennoufi A, Baumann N, Myelin-inducedexperimental allergic encephalomyelitis in Lewis rats: tumor necrosisfactor alpha levels in serum of cerebrospinal fluid immunohistochemicalexpression in glial cells and neurophages of optic nerve and spinalcord, J Neuroimmunol 1996; 64: 55-61; Wagner R, Myers R R, Endoneurialinjection of TNF-alpha produces neuropathic pain behaviours, Neuroreport1996; 7: 2897-901; Zhu J, Bai X F, Mix E, Link H, Cytokine dichotomy inperipheral nervous system influences the outcome of experimentalallergic neuritis: dynamics of MRNA expression for IL-1 beta, IL-6,IL-10, IL-12, TNF-alpha, TNF-beta, and cytolysin, Clin ImmunolImmunopathol 1997; 84: 85-94). TNF-alpha may also induce an increase invascular permeability (Redford E J, Hall S M, Smith K J, Vascularchances and demyclination induced by the intra neural injection oftumour necrosis factor, Brain 1995; 118: 869-78; Wagner R, Myers R R,Endoneurial injection of TNF-alpha produces neuropathic pain behaviours,Neuroreport 1996; 7: 2897-901) and initiate coagulation (Jurd K M,Stephens C J, Black M M, Hunt B J, Endothelial cell activation incutaneous vasculitis, Clin Exp Dermatol 1996; 21: 28-32; Nawroth P,Handley D, Matsueda G, De Waal R, Gerlach H, Blohm D, Stem D, Tumornecrosis factor/cachectin-induced intra vascular fibrin formation inmeth A fibrosarcomas, J Exp Med 1988; 168: 637-47; van der Poll T,Jansen P M, Van Zee K J, Welborn M Br, de Jong I, Hack C E, Loetscher H,Lesslauer W, Lowry S F, Moidawer L L, Tumor necrosis factor-alphainduces activation of coagulation and fibrinolysis in baboons through anexclusive effect on the p55 receptor, Blood 1996; 88: 922-7). Further,TNF-alpha may be blocked by steroids (Baumgartner R A, Deramo V A,Beaven M A, Constitutive and inducible mechanisms for synthesis andrelease of cytokines in immune cell lines, J Immunol 1996; 157: 4087-93;Brattsand R, Linden M, Cytokine modulation by glucocorticoids:mechanisms and actions in cellular studies, Aliment Pharmacol Ther 1996;10: 81-90; Iwamoto S, Takeda K, Possible cytotoxic mechanisms of TNF invitro, Hum Cell 1990; 3: 107-12; Teoh K H, Bradley C A, Galt J, BurrowsH, Steroid inhibition of cytokine-mediated vasodilation after warm heartsurgery, Circulation 1995; 92: 11347-53; Wershil B K, Furuta G T,Lavigne J A, Choudhury A R, Wang Z S, Galli S J, Dexamethasonecyclosporin A suppress mast cell-leukocyte cytokine cascades by multiplemechanisms, Int Arch Allergy Immunol 1995; 107: 323-4.), and cyclosporinA (Dawson J, Hurtenbach U, MacKenzie A, Cyclosporin A inhibits the invivo production of interleukin-Ibeta and tumour necrosis factor alpha,but not interleukin-6, by a T-cell independent mechanism, Cytokine 1996;8: 882-8; Smith C S, Ortega G, Parker L, Shearer WT, Cyclosporin Ablocks induction of tumor necrosis factor-alpha in human B lymphocytes,Biochem Biophys Res Commun 1994; 204: 383-90; Wasaki S, Sakaida I,Uchida K, Kiinura T, Kayano K, Oldta K, Preventive effect of cyclosporinA on experimentally induced acute liver injury in rats, Liver 1997; 17:107-14; Wershil B K, Furuta G T, Lavigne J A, Choudhury A R, Wang Z S,Galli S J, Dexamethasone cyclosporin A suppress mast cell-leukocytecytokine cascades by multiple mechanisms, Int Arch Allergy Immunol 1995;107: 323-4). However, the blocking effect on TNF-alpha is not sopronounced by NSAID (Garcia-Vicuna R, Diaz-Gonzalez F, Gonzalez-Alvaro1, del Pozo Ma, Mollinedo F, Cabanas C, Gonzalez-Amaro R, Sanchez-MadridF, Prevention of cytokine-induced changes in leucocyte adhesionreceptors by nonsteroidal antiinflammatory drugs from the oxicam family,Arthritis Rheum 1997; 40: 143-53; Gonzalez E, de la Cruz C, de NicolasR, Egido J, Herrero-Beaumont G, Long-term effect of nonsteroidalanti-inflammatory drugs on the production of cytokines and otherinflammatory mediators by blood cells of patients with osteosis, AgentsActions 1994; 41: 171-8; Herman J H, Sowder W G, Hess E V, Nonsteroidalantiinflammatory drug modulation of prosthesis pseudomembrane inducedbone resorption, J Rheunutol 1994; 21: 338-43) and very low or theagonized by lidocaine (Bidani A, Heming T A, Effects of lidocaine oncytosolic pH regulation and stimulus-induced effector functions inalveolar macrophages, Lung 1997; 175: 349-61; Matsumori A, Ono K, NishioR, Nose Y, Sasayaina S, Amiodarone inhibits production of tumor necrosisfactor-alpha by human mononuclear cells: a possible mechanism for itseffect in heart failure, Circulation 1997; 96: 1386 -9; Pichler W J,Zanni M, von Greyerz S, Schnyder B, Mauri-Hellweg D, Wendland, T, HighIL-5 production by human drug-specific T cell clones, Int Arch AllergyImmunol 1997; 113: 177-80; Takao Y, Mikawa K, Nishina Y, Maekawa N,Obara H, Lidocaine attenuates hyperoxic lung injury in rabbits, ActaAnaesthesiol Scand 1996; 40: 318-25).

[0127] It was recently observed that local application of nucleuspulposus may induce pain-related behavior in rats, particularly thermalhyperalgesia (Kawakami M, Tamaki T, Weinstein J N, Hashizume H, Nishi H,Meller S T, Pathomechanism of pain-related behaviour produced byallografts of intervertebral disc in the rat, Spine 1996; 21: 2101-7;Olmarker K, Myers R R, Pathogenesis of sciatic pain: Role of herniatednucleus pulposus and deformation of spinal nerve root and DRG, Pain,1998, 78: 9-105). TNF-alpha has also been found to be related to suchpain-behavioristic changes (DeLeo J A, Colbum R W, Rickman A J, Cytokineand growth factor immunohistochemical spinal profiles in two animalmodels of mononeuropathy, Brain Res 1997; 759: 50-7, Oka T, Wakugawa Y,Hosoi M, Oka K, Hari T, Intracerebroventricular injection of tumornecrosis factor-alpha induces thermal hyperalgesia in rats,Neuroimmunomodulation 1996; 3: 135-40; Sommer C, Schmidt C, George A,Toyka K V, A metalloprotease-inhibitor reduces pain associated behaviourin mice with experimental neuropathy, Neurosci Lett 1997; 237:45-8;Wagner R, Myers R R, Endoneurial injection of TNF-alpha producesneuropathic pain behaviours, Neuroreport 1996; 7: 2897-901), and also toneuropathies in general (Lin X H, Kashima Y, Khan M, Heller K B, Gu X Z,Sadun A A, An immunohistochemical study of TNF-alpha in optic nervesfrom AIDS patients, Curr Eye Res 1997; 16: 1064-8; Sharief M K, Ingram DA, Swash M, Circulating tumor necrosis factor-alpha correlates withelectrodiagnostic abnormalities in Guillain-Barre syndrome, Ann Neurol1997; 42: 6873; Sommer C, Schmidt C, George A, Toyka K V, Ametalloprotease-inhibitor reduces pain associated behaviour in mice withexperimental neuropathy, Neurosci Lett 1997; 237:45-8; Sorkin L S, XiaoW H, Wagner R, Myers R R, Tumour necrosis factor-alpha induces ectopicactivity in nociceptive primary afferent fibres, Neuroscience 1997; 81:255-62). However there are no studies that have assessed the possiblepresence of TNF-alpha in the cells of the nucleus pulposus.

[0128] To assess if TNF-alpha could be related to the observed nucleuspulposus induced reduction in nerve root conduction velocity it wasnecessary first to analyze if there was TNF-alpha in the nucleuspulposus-cells. The data clearly demonstrated that TNF-alpha was presentin these cells. TNF-alpha is produced as a precursor (pro-TNF) that isbound to the membrane, and it is activated by cleavage from thecell-membrane by a zinc-dependent metallo-endopeptidase (i.e., TNF-alphaconverting enzyme, TACE) (Black R A, Rauch C T, Kozlosky C J, Peschon IJ, Slack J L, Wolfson M F, Castner B J, Stocking K L, Reddy P,Srinivasan S, Nelson N, Boiani N, Schooley K A, Gerhart M, Davis R,Fitzner J N, Johnson R S, Paxton R J, March C J, Cerretti D P, Ametalloproteinase disintegrin that releases tumour-necrosis factor-alphafrom cells, Nature 1997; 385: 729-33; Gearing A J, Beckett P,Christodoulou M, Churchill M, Clements J, Davidson A H, Drummond A H,Galloway W A, Gilbert R, Gordon J L, et al., Processing of tumournecrosis factor-alpha precursor by metalloproteinases, Nature 1994; 370:555-7; Gazelle E J, Banda M J, Leppert D, Matrix metallo-proteinases inimmunity, J Immunol 1996; 156: 14; Robache-Gallea S, Bruneau J M, RobbeH, Morand V, Capdevila C, Bhatnagar N, Chouaib S, Roman-Roman S, Partialpurification and characterization of a tumor necrosis factor-alphaconverting activity, Eur J Immunol 1997; 27: 1275-82; Rosendahl M S, KoS C, Long D L, Brewer M T, Rosenzweig D, Hedl E, Anderson L, Pyle S M,Moreland J, Meyers M A, Kohno T, Lyons D, Lichenstein H S,Identification and characterization of a pro-tumor necrosisfactor-alpha-processing enzyme from the ADAM family of zincmetalloproteases, J Biol Chem 1997; 272: 24588-93). This may thus relatewell to experimental findings, where application of only thecell-membranes of autologous nucleus pulposus-cells induced nerveconduction velocity reduction, which indicated that the effects weremediated by a membrane-bound substance. Second, the effects of theTNF-alpha had to be blocked in a controlled manner. We then first choseto add the same selective antibody that was used forimmunohistochemistry in series 1, which is known to also block theeffects of TNF-alpha, to the nucleus pulposus before application. Also,we chose to treat the pigs with doxycycline, which is known to blockTNF-alpha (Kloppenburg M, B-an BM, de Rooij-Dijk H H, Mitenburg A M,Daha M R, Breedveld F C, Dijkmans B A, Verweij C, The tetracyclinederivative minocycline differentially affects cytokine production bymonocytes and T lymphocytes, Antimicrob Agents Chemother 1996; 40:934-40; Kloppenburg M, Verweij C L, Miltenburg A M, Verhoeven A J, DahaM R, Dikmans B A, Breeveld F C, The influence of tetracyclines on T cellactivation, Clin Exp. Immunol 1995; 102: 635-41; Milano S, Arcoleo F,D'Agostino P, Cillari E, Intraperitoneal injection of tetracyclinesprotects mice from lethal endotoxemia downregulating inducible nitricoxide synthase in various organs and cytokine and nitrate secretion inblood, Antimicrob Agents Chemother 1997; 41: 117-21; Shapira L, Houri Y,Barak V, Halabi A, Soskoine W A, Stabholz A, Human monocyte response tocementum extracts from periodontally diseased teeth: effect ofconditioning with tetracycline, J Periodontol 1996; 67: 682-7; ShapiraL, Houri Y, Barak V, Soskolne W A, Halabi A, Stabholz A, Tetracyclineinhibits Porphyromonas gingivalis lipopolysaccharide-induced lesions invivo and TNF processing in vitro, J Periodontal Res 1997; 32: 183-8).However, due to the low pH of the doxycycline preparation, it was chosento treat the pigs by intravenous injection instead of local addition tothe nucleus pulposus since nucleus pulposus at a low pH has been foundto potentiate the effects of the nucleus pulposus (Olmarker K, Byrod G,Comefjord M, Nordborg C, Rydevik B, Effects of methylprednisolone onnucleus pulposus-induced nerve root injury, Spine 1994; 19: 1803-8;Iwabuchi M, Rydevik B, Kikuchi S, Olmarker K, Methylprednisolone reducesthe early vascular permeability increase in spinal nerves by epiduralnucleus pulposus application, Accepted for publication in Spine).

[0129] Two recently developed drugs for specific TNF-alpha inhibitionwere also included in the study. Infliximab is a chimeric monoclonalantibody composed of human constant and murine variable regions.Infliximab binds specifically to human TNF-alpha. As opposed to themonoclonal antibody used in series-2 for the 3-day observation period,infliximab was not administered locally in the autotransplanted nucleuspulposus, but instead was administered systemically in a clinicallyrecommended dose (4 mg/kg).

[0130] Etanercept is a dimeric fusion protein consisting of the Fcportion of human IgG. The drug, etanercept, was administered in a dosagecomparable to the recommended dose for pediatric use (0.5 mg/kg, twice aweek).

[0131] The data regarding nerve conduction velocity showed that thereduction was completely blocked by the systemic-treatment and that thenerve conduction velocities in these series were close to the conductionvelocity after application of a control substance (retro peritoneal fat)from a previous study (Olmarker K, Rydevik B, Nordborg C, Autologousnucleus pulposus induces neurophysiologic and histologic changes inporcine cauda equina nerve roots, Spine 1993; 18: 1425-32). Applicationof the anti- TNF-alpha -antibody to the nucleus pulposus also partiallyprevented the reduction in nerve conduction velocity. However, thereduction was not as pronounced as that observed for doxycycline, andthe velocity in this series was not statistically different to thevelocity in the series with untreated animals, given the wide deviationof the data.

[0132] The local anti-TNF-alpha antibody treatment only partiallyblocked the nucleus pulposus-induced reduction of nerve conductionvelocity and the high standard deviation of the data could probably haveat least three different explanations. First, if looking at the specificdata within this group, it was found that the nerve conduction velocitywas low in 2 animals (mean 37.5 m/s) and high in 3 animals (mean 81.3m/s). There are thus 2 groups of distinctly different data within theanti- TNF-alpha treatment series. This will account for the highstandard deviation and might imply that the blocking effect wassufficient in 3 animals and insufficient in 2 animals. The lack ofeffects in these animals could be based simply on the amount ofantibodies in relation to TNF-alpha molecules not being sufficient, andif a higher dose of the antibody had been used, the TNF-alpha effectswould thus have been blocked even in these animals. Such a scenariocould then theoretically imply that TNF-alpha alone is responsible forthe observed nucleus pulposus-induced effects, and that this could notbe verified experimentally due to the amount of antibody being too low.

[0133] TNF-alpha may have various pathophysiologic effects. It may havedirect effects on tissues such as nerve tissue and blood vessels, it maytrigger other cells to produce other pathogenic substances and it maytrigger release of more TNF-alpha both by inflammatory cells and also bySchwann-cells locally in the nerve tissue (Wagner R, Myers R R, Schwanncells produce tumor necrosis factor alpha: expression in injurednon-injured nerves, Neuroscience 1996; 73: 625-9). There is thus reasonto believe that even low amounts of TNF-alpha may be sufficient toinitiate these processes and that there is a local recruitment ofcytokine producing cells and a subsequent increase in production andrelease of other cytokines as well as TNF-alpha. TNF-alpha may thereforeact as the “ignition key” of the pathophysiologic processes and play animportant role for the initiation of the pathophysiologic cascade behindthe nucleus pulposus-induced nerve injury. However, the majorcontribution of TNF-alpha may be derived from recruited, aggregated andmaybe even extravasated leukocytes, and that successful pharmacologicblock may be achieved only by systemic treatment.

[0134] In conclusion, for the first time a specific substance(TNF-alpha) has been linked to the nucleus pulposus-induced nerve rootinjury. This new information may be of significant importance for thecontinued understanding of nucleus pulposus-induced nerve injury as wellas raising the question of the potential future clinical use ofpharmacological interference with TNF-alpha and related substances, fortreatment of sciatica.

[0135] The presence of TNF-alpha in pig nucleus pulposus-cells was thusimmunohistochemically verified. Block of TNF-alpha by a locally appliedmonoclonal antibody limited the nucleus pulposus-induced reduction ofnerve root conduction velocity, whereas intravenous treatment withdoxycycline, infliximab, and etanercept significantly blocked thisreduction. These data for the first time links one specific substance,TNF-alpha, to the nucleus pulposus-induced nerve injury.

Example 3

[0136] CDP-571 (HUMICADE®)

[0137] A 43-year old man with radiating pain corresponding to the left4th lumbar nerve root is diagnosed as having sciatica with nerve rootdisturbance. He will be treated with 10 mg/kg of CDP-571 (HUMICADE®)intravenously in a single dose.

Example 4

[0138] D2E7

[0139] A 38-year old female with radiating pain and slight nervedysfunction corresponding to the 1st sacral nerve on the left side isdiagnosed as having a disc herniation with sciatica. She will be treatedwith an intravenous injection of 5 mg/kg of D2E7.

Example 5

[0140] CDP-870

[0141] A 41-year old female with dermatomal pain corresponding to thefirst sacral nerve root on the left side is examined revealing noneurological deficit but a positive straight leg raising test on theleft side. She will be treated with an intravenous injection of 5 mg/kgof CDP-870.

Example 6

[0142] Combination treatment with a TNF inhibitor and an IL-1 inhibitor

[0143] Fourteen pigs, (body weight 25-30 kg) received an intramuscularinjection of 20 mg/kg body weight of KETALAR® (ketamine 50 mg/ml,Parke-Davis, Morris Plains, N.J.) and an intravenous injection of 4mg/kg body weight of HYPNODIL® (methomidate chloride 50 mg/ml, AB Leo,Helsingborg, Sweden) and 0.1 mg/kg body weight of STRESNIL® (azaperon 2mg/ml, Janssen Pharmaceutica, Beerse, Belgium). Anesthesia wasmaintained by additional intravenous injections of 2 mg/kg body weightof HYPNODIL® and 0.05 mg/kg body weight of STRESNIL®. The pigs alsoreceived an intravenous injection of 0.1 mg/kg of STESOLID NOVUM®(Diazepam, Dumex, Helsingborg) after surgery.

[0144] Nucleus pulposus was harvested from the 5th lumbar disc through aretroperitoneal approach. Approximately 40 mg of the nucleus pulposuswas applied to the sacrococcygeal cauda equina through a midlineincision and laminectomy of the first coccygeal vertebra. In 5 pigs, thenucleus pulposus was mixed with 100 μg of an anti-TNF-alpha antibody(anti-pig TNF-alpha monoclonal purified antibody, Endogen, Woburn,Mass., USA, Ordering Code MP-390) before application. In five pigs, thenucleus pulposus was mixed with both 100 μg of an anti-TNF-alphaantibody and 100 μg of an anti-IL-1β antibody (anti-pig IL-1β monoclonalpurified antibody, Endogen, Woburn, Mass., USA, Catalog No. MP-425). Inthe remaining 4 pigs, 2 mg of doxycycline (DOXYFERM®, doxycycline 20mg/ml, Nordic, Malmö, Sweden) was mixed with the nucleus pulposus beforeapplication.

[0145] Three days after the application, the pigs were reanaesthetizedby an intramuscular injection of 20 mg/kg body weight of KETALAR® and anintravenous injection of 35 mg/kg body weight of PENTOTHAL® (Thiopentalsodium, Abbott lab, Chicago, Ill.). The pigs were ventilated on arespirator. Anesthesia was maintained by an intravenous bolus injectionof 100 mg/kg body weight of Chloralose (alpha)-D(+)-gluco-chloralose,Merck, Darmstadt, Germany) and by a continuous supply of 30 mg/kg/hourof Chloralose. A laminectomy from the 4th sacral to the 3rd coccygealvertebra was performed. The nerve roots were covered with SPONGOSTANE®(Ferrosan, Denmark). Local tissue temperature was continuously monitoredand maintained at 37.5-38.0° C. by means of a heating lamp.

[0146] The cauda equina was stimulated by two E2 subdermal platinumneedle electrodes (Grass Instrument Co., Quincy, Mass.) which wereconnected to a Grass SD9 stimulator (Grass Instrument Co., Quincy,Mass.) and gently placed intermittently on the cauda equina first 10 mmcranial and then 10 mm caudal to the exposed area. To ensure that onlyimpulses from exposed nerve fibers were registered, the nerve root thatexited from the spinal canal between the two stimulation sites were cut.An EMG was registered by two subdermal platinum needle electrodes, whichwere placed into the paraspinal muscles in the tail approximately 10 mmapart. This procedure is reproducible and represents a functionalmeasurement of the motor nerve fibers of the cauda equina nerve roots.The EMG was visualized using a Macintosh IIci computer provided withSuperscope software and MacAdios II A/D converter (GW Instruments,Sommerville, Mass.). The separation distance between the first peaks ofthe EMG from the two recordings was determined and the separationdistance between the two stimulation sites on the cauda equina wasmeasured with calipers. The nerve conduction velocity between the twostimulation sites could thus be calculated from these two measurements.

[0147] The person performing the neurophysiologic analyzes was unawareof the experimental protocol for the individual animal, and afterfinishing the complete study the data were arranged in the threeexperimental groups and statistical differences between the groups wereassessed by Student's t-test. For comparison, data from a previous studyof the effects of application of retroperitoneal fat and autologousnucleus pulposus were included (Olmarker K, Rydevik B, Nordborg C,Autologous nucleus pulposus induces neurophysiologic and histologicchanges in porcine cauda equina nerve roots, Spine 1993; 18(11):1425-32).

[0148] The average nerve conduction velocity for the five groups isdisplayed in Table 3 below. According to the previous study, baselinenerve conduction velocity (fat) was 83 m/s and application of autologousnucleus pulposus induced a reduction of the conduction velocity to 45m/s (Olmarker K, Rydevik B, Nordborg C, Autologous nucleus pulposusinduces neurophysiologic and histologic changes in porcine cauda equinanerve roots, Spine 1993; 18(11): 1425-32). Application of doxycycline tothe nucleus pulposus reduced the effects of the nucleus pulposus.Addition of an anti-TNF antibody was more efficient in reducing thenucleus pulposus effects than doxycycline. However, most efficient inblocking the nucleus pulposus induced reduction in nerve conductionvelocity was the combined action of the anti-TNF and the anti-IL-1antibody. TABLE 3 Nerve conduction velocity (m/s) 3 days afterapplication Fat (n = 5) 83 ± 4  NP + doxycycline (n = 4) 53 ± 22 NP +anti-TNF (n = 5) 64 ± 28 NP + anti-TNF and anti-IL-1β (n = 5) 74 ± 2  NP(n = 5) 45 ± 16

Example 7

[0149] Combination treatment with a TNF inhibitor and an IL-1 inhibitor

[0150] Seven pigs, (body weight 25-30 kg) received an intramuscularinjection of 20 mg/kg body weight of KETALAR® (ketamine 50 mg/ml,Parke-Davis, Morris Plains, N.J.) and an intravenous injection of 4mg/kg body weight of HYPNODIL® (methomidate chloride 50 mg/ml, AB Leo,Helsingborg, Sweden) and 0.1 mg/kg body weight of STRESNIL® (azaperon 2mg/ml, Janssen Pharmaceutica, Beerse, Belgium). Anesthesia wasmaintained by additional intravenous injections of 2 mg/kg body weightof HYPNODIL® and 0.05 mg/kg body weight of STRESNIL®. The pigs alsoreceived an intravenous injection of 0.1 mg/kg of STESOLID NOVUM®(Diazepam, Dumex, Helsingborg) after surgery.

[0151] Nucleus pulposus was harvested from the 5th lumbar disc through aretroperitoneal approach. Approximately 40 mg of the nucleus pulposuswas applied to the sacrococcygeal cauda equina through a midlineincision and laminectomy of the first coccygeal vertebra. In 2 pigs, thenucleus pulposus was mixed with 100 μg of an anti-TNF-alpha antibody(anti-pig TNF-alpha monoclonal purified antibody, Endogen, Woburn,Mass., USA, Ordering Code MP-390) before application. In 2 pigs, thenucleus pulposus was mixed with 100 μg of an anti-IL-1β antibody(anti-pig IL-1β monoclonal purified antibody, Endogen, Woburn, Mass.,USA, Catalog No. MP-425). In five pigs, the nucleus pulposus was mixedwith both 100 μg of an anti-TNF-alpha antibody and 100 μg of ananti-IL-1β antibody.

[0152] Seven days after the application, the pigs were reanaesthetizedby an intramuscular injection of 20 mg/kg body weight of KETALAR® and anintravenous injection of 35 mg/kg body weight of PENTOTHAL® (Thiopentalsodium, Abbott lab, Chicago, Ill.). The pigs were ventilated on arespirator. Anesthesia was maintained by an intravenous bolus injectionof 100 mg/kg body weight of Chloralose (alpha)-D(+)-gluco-chloralose,Merck, Darmstadt, Germany) and by a continuous supply of 30 mg/kg/hourof Chloralose. A laminectomy from the 4th sacral to the 3rd coccygealvertebra was performed. The nerve roots were covered with SPONGOSTANE®(Ferrosan, Denmark). Local tissue temperature was continuously monitoredand maintained at 37.5-38.0° C. by means of a heating lamp.

[0153] The cauda equina was stimulated by two E2 subdermal platinumneedle electrodes (Grass Instrument Co., Quincy, Mass.) which wereconnected to a Grass SD9 stimulator (Grass Instrument Co., Quincy,Mass.) and gently placed intermittently on the cauda equina first 10 mmcranial and then 10 mm caudal to the exposed area. To ensure that onlyimpulses from exposed nerve fibers were registered, the nerve root thatexited from the spinal canal between the two stimulation sites were cut.An EMG was registered by two subdermal platinum needle electrodes, whichwere placed into the paraspinal muscles in the tail approximately 10 mmapart. This procedure is reproducible and represents a functionalmeasurement of the motor nerve fibers of the cauda equina nerve roots.The EMG was visualized using a Macintosh IIci computer provided withSuperscope software and MacAdios II A/D converter (GW Instruments,Sommerville, Mass.). The separation distance between the first peaks ofthe EMG from the two recordings was determined and the separationdistance between the two stimulation sites on the cauda equina wasmeasured with calipers. The nerve conduction velocity between the twostimulation sites could thus be calculated from these two measurements.

[0154] The person performing the neurophysiologic analyzes was unawareof the experimental protocol for the individual animal, and afterfinishing the complete study the data were arranged in the threeexperimental groups and statistical differences between the groups wereassessed by Student's t-test. The local animal research ethics committeeapproved the experimental protocol for this experiment. For comparison,data from a previous study of the effects of application ofretroperitoneal fat and autologous nucleus pulposus were included(Olmarker K, Rydevik B, Nordborg C, Autologous nucleus pulposus inducesneurophysiologic and histologic changes in porcine cauda equina nerveroots, Spine 1993; 18(11): 1425-32). The average nerve conductionvelocity for the five groups is displayed in the Table 4 below.According to the previous study, baseline nerve conduction velocity(fat) was 76 m/s and application of autologous nucleus pulposus induceda reduction of the conduction velocity to 45 m/s (Olmarker K, Rydevik B,Nordborg C, Autologous nucleus pulposus induces neurophysiologic andhistologic changes in porcine cauda equina nerve roots, Spine 1993;18(11): 1425-32). Application of anti IL-1 showed a similar NCV as NPalone, indicating that the IL-1 had could not reduce the nucleuspulposus-induced reduction in nerve conduction velocity. Application ofanti TNF limited the nucleus pulposus-induced reduction in nerveconduction velocity. Most efficient in reducing the nucleuspulposus-induced reduction in nerve conduction velocity was thecombination of anti IL-1 and anti TNF. Since the effect in limiting thereduction in NCV was more than could be anticipated for addition of theeffects of anti IL-1 and anti TNF, the data indicate a synergisticeffect of the combination of the two cytokine antagonists. TABLE 4 Nerveconduction velocity (m/s) 7 days after application Fat (n = 5) 76 ± 11NP + anti-IL-1β (n = 2) 46 ± 11 NP + anti-TNF (n = 2) 59 ± 6  NP +anti-TNF and anti-IL-1β (n = 3) 78 ± 2  NP (n = 5) 45 ± 19

[0155] The amplitude of the obtained muscle action potentials (MAP's)grossly reflects the number of conducting axons. The amplitudes afterapplication of the cytokine inhibitors were measured and compared toprevious data of application of annulus fibrosus (NCV 75±11 m/s) andnucleus pulposus at ph 6.0 (NCV 55±22 m/s) from a previous study(Iwabuchi M, Rydevik B, Kikuchi S, Olmarker K, Effects of anulusfibrosus and experimentally degenerated nucleus pulposus on nerve rootconduction velocity, Spine 2001; 26(15): 1651-5) and presented in table5. Amplitudes of MAP seven days after application of cultured nucleuspulposus cells (NCV 52±14 m/s) were also used as reference and exampleof the MAP amplitude in NP-exposed nerve roots (Kayama S, Olmarker K,Larsson K, Sjögren-Jansson E, Lindahl A, Cultured, autologous nucleuspulposus cells induce functional changes in spinal nerve roots, Spine1998; 23(20): 2155-8). NP at pH 6.0 was obtained by slightly loweringthe pH to 6.0 by addition of sodium lactate to form a 15 mmol/L lactateconcentration and to adjusting the pH to 6.0 by adding a 2% HClsolution. The nerve recordings in the NP at pH 6.0 series were performed3 days after application. The difference between application of NP at6.0 and the combination of an anti-TNF and an anti-IL-1 inhibitor wasstatistically significant using Students t-test (p=0.0384), whereas thedifference between NP at 6.0 and the two inhibitors alone were not.Similar, the difference between application of NP cells and thecombination of an anti-TNF and an anti-IL-1 inhibitor was statisticallysignificant using Students t-test (p=0.0426), whereas the differencebetween NP cells and the two inhibitors alone were not. Since the effectin limiting the MAP amplitude was more than could be anticipated foraddition of the effects of anti IL-1 and anti TNF, the data regardingMAP amplitude also indicate a synergistic effect of the combination ofthe two cytokine antagonists. TABLE 5 Muscle action potential amplitude(mVolt) Annulus fibrosus (n = 5) 6.5 ± 2.6 NP + anti-IL-1β (n = 2) 2.0 ±1.8 NP + anti-TNF (n = 2) 4.1 ± 0.4 NP + anti-TNF and anti-IL-1β (n = 3)6.1 ± 2.6 NP at pH 6.0 (n = 5) 2.9 ± 1.0 NP cells (n = 5) 2.8 ± 1.2

1. A method for treatment of a nerve disorder in a subject comprisingadministering to said subject a therapeutically effective dosage of atleast two substances selected from the group consisting of TNFinhibitors, IL-1 inhibitors, IL-6 inhibitors, IL-8 inhibitors, FASinhibitors, FAS ligand inhibitors, and IFN-gamma inhibitors.
 2. Themethod of claim 1, wherein one of said at least two substances is an MMPinhibitor.
 3. The method of claim 2, wherein said MMP inhibitor isdoxycycline.
 4. The method of claim 1, wherein one of said at least twosubstances is an IL-1 inhibitor.
 5. The method of claim 4, wherein saidIL-1 inhibitor is anakinra.
 6. The method of claim 1, wherein thesubject is a vertebrate.
 7. The method of claim 6, wherein thevertebrate is a mammal.
 8. The method of claim 7, wherein the mammal isa human.
 9. The method of claim 1, wherein said nerve disorder is due toa reduced nerve reduction velocity.
 10. The method of claim 1, whereinsaid nerve disorder is a spinal disorder.
 11. The method of claim 1,wherein said nerve disorder is nerve root injury.
 12. The method ofclaim 1, wherein said nerve disorder is caused by a disk herniation. 13.The method of claim 1, wherein said nerve disorder is sciatica.
 14. Themethod of claim 1, wherein said disorder is low back pain.
 15. Themethod of claim 1, wherein said disorder is whiplash associateddisorder.
 16. The method of claim 1, wherein said nerve disorderinvolves pain.
 17. The method of claim 1, wherein said nerve disorder isa nucleus pulposus-induced nerve injury.
 18. The method of claim 1,wherein said disorder is cervical rhizopathy.
 19. The method of claim 1,wherein said nerve disorder is spinal cord compression.
 20. The methodof claim 1, wherein said substances are administered systemically orlocally.
 21. The method of claim 1, wherein said substances areadministered parenterally.
 22. The method of claim 1, wherein saidsubstances are administered intramuscularly, intravenously,subcutaneously, orally, or rectally.
 23. The method of claim 22, whereinsaid substances are administered intravenously by injection or infusion.24. A method for treatment of a nerve disorder in a subject comprisingadministering to said subject a therapeutically effective dosage of aTNF inhibitor in combination with a second inhibitor selected from thegroup consisting of IL-1 inhibitors, IL-6 inhibitors, IL-8 inhibitors,FAS inhibitors, FAS ligand inhibitors, and IFN-gamma inhibitors.
 25. Themethod of claim 24, wherein said TNF inhibitor is infliximab, CDP-571(HUMICADE™), D2E7, or CDP-870.
 26. The method of claim 24, wherein saidTNF inhibitor is a soluble cytokine TNF receptor.
 27. The method ofclaim 26, wherein said soluble cytokine TNF receptor is etanercept. 28.The method of claim 24, wherein said TNF inhibitor is a binuclear DNAthreading transition metal complex with anti-cancer effect.
 29. Themethod of claim 24, wherein said TNF inhibitor is a lactoferrinderivable peptide.
 30. The method of claim 24, wherein one of said atleast two substances is an MMP inhibitor.
 31. The method of claim 30,wherein said MMP inhibitor is doxycycline.
 32. The method of claim 24,wherein said TNF inhibitor is a p38 kinase inhibitor.
 33. The method ofclaim 24, wherein said TNF inhibitor is TTP.
 34. The method of claim 24,wherein the subject is a vertebrate.
 35. The method of claim 34, whereinthe vertebrate is a mammal.
 36. The method of claim 35, wherein themammal is a human.
 37. The method of claim 24, wherein said nervedisorder is due to a reduced nerve reduction velocity.
 38. The method ofclaim 24, wherein said nerve disorder is a spinal disorder.
 39. Themethod of claim 24, wherein said nerve disorder is nerve root injury.40. The method of claim 24, wherein said nerve disorder is caused by adisk herniation.
 41. The method of claim 24, wherein said nerve disorderis sciatica.
 42. The method of claim 24, wherein said disorder is lowback pain.
 43. The method of claim 24, wherein said disorder is whiplashassociated disorder.
 44. The method of claim 24, wherein said nervedisorder involves pain.
 45. The method of claim 24, wherein said nervedisorder is a nucleus pulposus-induced nerve injury.
 46. The method ofclaim 24, wherein said disorder is cervical rhizopathy.
 47. The methodof claim 24, wherein said nerve disorder is spinal cord compression. 48.The method of claim 24, wherein said substances are administeredsystemically or locally.
 49. The method of claim 24, wherein saidsubstances are administered parenterally.
 50. The method of claim 24,wherein said substances are administered intramuscularly, intravenously,subcutaneously, orally, or rectally.
 51. The method of claim 50, whereinsaid substances are administered intravenously by injection or infusion.52. The method of claim 51, wherein said TNF inhibitor is administeredorally at a dosage of about 20 mg to about 1,500 mg.
 53. The method ofclaim 24, wherein said TNF inhibitor is D2E7 and is administered in adosage of about 0.1 mg/kg to about 50 mg/kg body weight of said subject.54. The method of claim 24, wherein said TNF inhibitor is CDP-870 and isadministered in a dosage of about 1 mg/kg to about 50 mg/kg body weightof said subject.
 55. A method for treatment of a nerve disorder in asubject comprising administering to said subject a therapeuticallyeffective dosage of a first TNF inhibitor in combination with a secondTNF inhibitor.
 56. The method of claim 55, wherein said first TNFinhibitor is a specific TNF inhibitor and said second TNF inhibitor is anon-specific TNF inhibitor.
 57. The method of claim 56, wherein saidspecific TNF inhibitor is infliximab, CDP-571 (HUMICADE™), D2E7, orCDP-870.
 58. The method of claim 56, wherein said specific TNF inhibitoris a soluble cytokine TNF receptor.
 59. The method of claim 58, whereinsaid soluble cytokine TNF receptor is etanercept.
 60. The method ofclaim 56, wherein said non-specific TNF inhibitor is a TNF inhibitor inthe form of a binuclear DNA threading transition metal complex withanti-cancer effect.
 61. The method of claim 56, wherein saidnon-specific TNF inhibitor is a TNF inhibitor in the form of alactoferrin derivable peptide.
 62. The method of claim 56, wherein saidnon-specific TNF inhibitor is an MMP inhibitor.
 63. The method of claim62, wherein said MMP inhibitor is doxycycline.
 64. The method of claim56, wherein said non-specific TNF inhibitor is a p38 kinase inhibitor.65. The method of claim 56, wherein said non-specific TNF inhibitor isTTP.
 66. The method of claim 56, wherein the subject is a vertebrate.67. The method of claim 66, wherein the vertebrate is a mammal.
 68. Themethod of claim 67, wherein the mammal is a human.
 69. The method ofclaim 56, wherein said nerve disorder is due to a reduced nervereduction velocity.
 70. The method of claim 56, wherein said nervedisorder is a spinal disorder.
 71. The method of claim 56, wherein saidnerve disorder is nerve root injury.
 72. The method of claim 56, whereinsaid nerve disorder is caused by a disk herniation.
 73. The method ofclaim 56, wherein said nerve disorder is sciatica.
 74. The method ofclaim 56, wherein said disorder is low back pain.
 75. The method ofclaim 56, wherein said disorder is whiplash associated disorder.
 76. Themethod of claim 56, wherein said nerve disorder involves pain.
 77. Themethod of claim 56, wherein said nerve disorder is a nucleuspulposus-induced nerve injury.
 78. The method of claim 56, wherein saiddisorder is cervical rhizopathy.
 79. The method of claim 56, whereinsaid nerve disorder is spinal cord compression.
 80. The method of claim56, wherein said substances are administered systemically or locally.81. The method of claim 56, wherein said substances are administeredparenterally.
 82. The method of claim 56, wherein said substances areadministered intramuscularly, intravenously, subcutaneously, orally, orrectally.
 83. The method of claim 82, wherein said substances areadministered intravenously by injection or infusion.
 84. The method ofclaim 83, wherein said specific TNF inhibitor is administered orally ata dosage of about 20 mg to about 1,500 mg.
 85. The method of claim 56,wherein said specific TNF inhibitor is D2E7 and is administered in adosage of about 0.1 mg/kg to about 50 mg/kg body weight of said subject.86. The method of claim 56, wherein said specific TNF inhibitor isCDP-870 and is administered in a dosage of about 1 mg/kg to about 50mg/kg body weight of said subject.
 87. A method for treatment of a nervedisorder in a subject comprising administering to said subject atherapeutically effective dosage of a TNF inhibitor in combination witha IL-1 inhibitor.
 88. The method of claim 87, wherein said TNF inhibitoris infliximab, CDP-571 (HUMICADE™), D2E7, or CDP-870.
 89. The method ofclaim 87, wherein said TNF inhibitor is a soluble cytokine TNF receptor.90. The method of claim 87, wherein said soluble cytokine TNF receptoris etanercept.
 91. The method of claim 87, wherein said TNF inhibitor isa binuclear DNA threading transition metal complex with anti-cancereffect.
 92. The method of claim 87, wherein said TNF inhibitor is alactoferrin derivable peptide.
 93. The method of claim 87, wherein oneof said at least two substances is an MMP inhibitor.
 94. The method ofclaim 93, wherein said MMP inhibitor is doxycycline.
 95. The method ofclaim 87, wherein said TNF inhibitor is a p38 kinase inhibitor.
 96. Themethod of claim 87, wherein said TNF inhibitor is TTP.
 97. The method ofclaim 87, wherein said IL-1 inhibitor is anakinra.
 98. The method ofclaim 87, wherein the subject is a vertebrate.
 99. The method of claim98, wherein the vertebrate is a mammal.
 100. The method of claim 99,wherein the mammal is a human.
 101. The method of claim 87, wherein saidnerve disorder is due to a reduced nerve reduction velocity.
 102. Themethod of claim 87, wherein said nerve disorder is a spinal disorder.103. The method of claim 87, wherein said nerve disorder is nerve rootinjury.
 104. The method of claim 87, wherein said nerve disorder iscaused by a disk herniation.
 105. The method of claim 87, wherein saidnerve disorder is sciatica.
 106. The method of claim 87, wherein saiddisorder is low back pain.
 107. The method of claim 87, wherein saiddisorder is whiplash associated disorder.
 108. The method of claim 87,wherein said nerve disorder involves pain.
 109. The method of claim 87,wherein said nerve disorder is a nucleus pulposus-induced nerve injury.110. The method of claim 87, wherein said disorder is nerve root injury.111. The method of claim 87, wherein said nerve disorder is spinal cordcompression.
 112. The method of claim 87, wherein said substances areadministered systemically or locally.
 113. The method of claim 87,wherein said substances are administered parenterally.
 114. The methodof claim 87, wherein said substances are administered intramuscularly,intravenously, subcutaneously, orally, or rectally.
 115. The method ofclaim 114, wherein said substances are administered intravenously byinjection or infusion.
 116. The method of claim 115, wherein said TNFinhibitor is administered orally at a dosage of about 20 mg to about1,500 mg.
 117. The method of claim 87, wherein said TNF inhibitor isD2E7 and is administered in a dosage of about 0.1 mg/kg to about 50mg/kg body weight of said subject.
 118. The method of claim 87, whereinsaid TNF inhibitor is CDP-870 and is administered in a dosage of about 1mg/kg to about 50 mg/kg body weight of said subject.
 119. Apharmaceutical composition for treatment of a nerve disorder incomprising a therapeutically effective dosage of at least two substancesselected from the group consisting of TNF inhibitors, IL-1 inhibitors,IL-6 inhibitors, IL-8 inhibitors, FAS inhibitors, FAS ligand inhibitors,and IFN-gamma inhibitors.
 120. The pharmaceutical composition of claim119, wherein one of said at least two substances is an MMP inhibitor.121. The pharmaceutical composition of claim 119, wherein said MMPinhibitor is doxycycline.
 122. The pharmaceutical composition of claim119, wherein one of said at least two substances is an IL-1 inhibitor.123. The pharmaceutical composition of claim 122, wherein said IL-1inhibitor is anakinra.
 124. The pharmaceutical composition of claim 119,wherein said nerve disorder is due to a reduced nerve reductionvelocity.
 125. The pharmaceutical composition of claim 119, wherein saidnerve disorder is a spinal disorder.
 126. The pharmaceutical compositionof claim 119, wherein said nerve disorder is nerve root injury.
 127. Thepharmaceutical composition of claim 119, wherein said nerve disorder iscaused by a disk herniation.
 128. The pharmaceutical composition ofclaim 119, wherein said nerve disorder is sciatica.
 129. Thepharmaceutical composition of claim 119, wherein said disorder is lowback pain.
 130. The pharmaceutical composition of claim 119, whereinsaid disorder is whiplash associated disorder.
 131. The pharmaceuticalcomposition of claim 119, wherein said nerve disorder involves pain.132. The pharmaceutical composition of claim 119, wherein said nervedisorder is a nucleus pulposus-induced nerve injury.
 133. Thepharmaceutical composition of claim 119, wherein said disorder iscervical rhizopathy.
 134. The pharmaceutical composition of claim 119,wherein said nerve disorder is spinal cord compression.
 135. Apharmaceutical composition for treatment of a nerve disorder comprisinga therapeutically effective dosage of a TNF inhibitor in combinationwith a second inhibitor selected from the group consisting of IL-1inhibitors, IL-6 inhibitors, IL-8 inhibitors, FAS inhibitors, FAS ligandinhibitors, and IFN-gamma inhibitors.
 136. The pharmaceuticalcomposition of claim 135, wherein said TNF inhibitor is infliximab,CDP-571 (HUMICADE™), D2E7, or CDP-870.
 137. The pharmaceuticalcomposition of claim 135, wherein said TNF inhibitor is a solublecytokine TNF receptor.
 138. The pharmaceutical composition of claim 137,wherein said soluble cytokine TNF receptor is etanercept.
 139. Thepharmaceutical composition of claim 135, wherein said TNF inhibitor is abinuclear DNA threading transition metal complex with anti-cancereffect.
 140. The pharmaceutical composition of claim 135, wherein saidTNF inhibitor is a lactoferrin derivable peptide.
 141. Thepharmaceutical composition of claim 135, wherein one of said at leasttwo substances is an MMP inhibitor.
 142. The pharmaceutical compositionof claim 141, wherein said MMP inhibitor is doxycycline.
 143. Thepharmaceutical composition of claim 135, wherein said TNF inhibitor is ap38 kinase inhibitor.
 144. The pharmaceutical composition of claim 135,wherein said TNF inhibitor is TTP.
 145. The pharmaceutical compositionof claim 135, wherein said nerve disorder is due to a reduced nervereduction velocity.
 146. The pharmaceutical composition of claim 135,wherein said nerve disorder is a spinal disorder.
 147. Thepharmaceutical composition of claim 135, wherein said nerve disorder isnerve root injury.
 148. The pharmaceutical composition of claim 135,wherein said nerve disorder is caused by a disk herniation.
 149. Thepharmaceutical composition of claim 135, wherein said nerve disorder issciatica.
 150. The pharmaceutical composition of claim 135, wherein saiddisorder is low back pain.
 151. The pharmaceutical composition of claim135, wherein said disorder is whiplash associated disorder.
 152. Thepharmaceutical composition of claim 135, wherein said nerve disorderinvolves pain.
 153. The pharmaceutical composition of claim 135, whereinsaid nerve disorder is a nucleus pulposus-induced nerve injury.
 154. Thepharmaceutical composition of claim 135, wherein said disorder iscervical rhizopathy.
 155. The pharmaceutical composition of claim 135,wherein said nerve disorder is spinal cord compression.
 156. Apharmaceutical composition for treatment of a nerve disorder comprisinga therapeutically effective dosage of a first TNF inhibitor incombination with a second TNF inhibitor.
 157. The pharmaceuticalcomposition of claim 156, wherein said first TNF inhibitor is a specificTNF inhibitor and said second TNF inhibitor is a non-specific TNFinhibitor.
 158. The pharmaceutical composition of claim 157, whereinsaid specific TNF inhibitor is infliximab, CDP-571 (HUMICADE™), D2E7, orCDP-870.
 159. The pharmaceutical composition of claim 157, wherein saidspecific TNF inhibitor is a soluble cytokine TNF receptor.
 160. Thepharmaceutical composition of claim 159, wherein said soluble cytokineTNF receptor is etanercept.
 161. The pharmaceutical composition of claim157, wherein said non-specific TNF inhibitor is a TNF inhibitor in theform of a binuclear DNA threading transition metal complex withanti-cancer effect.
 162. The pharmaceutical composition of claim 157,wherein said non-specific TNF inhibitor is a TNF inhibitor in the formof a lactoferrin derivable peptide.
 163. The pharmaceutical compositionof claim 157, wherein said non-specific TNF inhibitor is an MMPinhibitor.
 164. The pharmaceutical composition of claim 163, whereinsaid MMP inhibitor is doxycycline.
 165. The pharmaceutical compositionof claim 157, wherein said non-specific TNF inhibitor is a p38 kinaseinhibitor.
 166. The pharmaceutical composition of claim 157, whereinsaid non-specific TNF inhibitor is TTP.
 167. The pharmaceuticalcomposition of claim 157, wherein said nerve disorder is due to areduced nerve reduction velocity.
 168. The pharmaceutical composition ofclaim 157, wherein said nerve disorder is a spinal disorder.
 169. Thepharmaceutical composition of claim 157, wherein said nerve disorder isnerve root injury.
 170. The pharmaceutical composition of claim 157,wherein said nerve disorder is caused by a disk herniation.
 171. Thepharmaceutical composition of claim 157, wherein said nerve disorder issciatica.
 172. The pharmaceutical composition of claim 157, wherein saiddisorder is low back pain.
 173. The pharmaceutical composition of claim157, wherein said disorder is whiplash associated disorder.
 174. Thepharmaceutical composition of claim 157, wherein said nerve disorderinvolves pain.
 175. The pharmaceutical composition of claim 157, whereinsaid nerve disorder is a nucleus pulposus-induced nerve injury.
 176. Thepharmaceutical composition of claim 157, wherein said disorder iscervical rhizopathy.
 177. The pharmaceutical composition of claim 157,wherein said nerve disorder is spinal cord compression.
 178. Apharmaceutical composition for treatment of a nerve disorder comprisinga therapeutically effective dosage of a TNF inhibitor in combinationwith an IL-1 inhibitor.
 179. The pharmaceutical composition of claim178, wherein said TNF inhibitor is infliximab, CDP-571 (HUMICADE™),D2E7, or CDP-870.
 180. The pharmaceutical composition of claim 178,wherein said TNF inhibitor is a soluble cytokine TNF receptor.
 181. Thepharmaceutical composition of claim 180, wherein said soluble cytokineTNF receptor is etanercept.
 182. The pharmaceutical composition of claim178, wherein said TNF inhibitor is a binuclear DNA threading transitionmetal complex with anti-cancer effect.
 183. The pharmaceuticalcomposition of claim 178, wherein said TNF inhibitor is a lactoferrinderivable peptide.
 184. The pharmaceutical composition of claim 178,wherein one of said at least two substances is an MMP inhibitor. 185.The pharmaceutical composition of claim 184, wherein said MMP inhibitoris doxycycline.
 186. The pharmaceutical composition of claim 178,wherein said TNF inhibitor is a p38 kinase inhibitor.
 187. Thepharmaceutical composition of claim 178, wherein said TNF inhibitor isTTP.
 188. The pharmaceutical composition of claim 178, wherein said IL-1inhibitor is anakinra.
 189. The pharmaceutical composition of claim 178,wherein said nerve disorder is due to a reduced nerve reductionvelocity.
 190. The pharmaceutical composition of claim 178, wherein saidnerve disorder is a spinal disorder.
 191. The pharmaceutical compositionof claim 178, wherein said nerve disorder is nerve root injury.
 192. Thepharmaceutical composition of claim 178, wherein said nerve disorder iscaused by a disk herniation.
 193. The pharmaceutical composition ofclaim 178, wherein said nerve disorder is sciatica.
 194. Thepharmaceutical composition of claim 178, wherein said disorder is lowback pain.
 195. The pharmaceutical composition of claim 178, whereinsaid disorder is whiplash associated disorder.
 196. The pharmaceuticalcomposition of claim 178, wherein said nerve disorder involves pain.197. The pharmaceutical composition of claim 178, wherein said nervedisorder is a nucleus pulposus-induced nerve injury.
 198. Thepharmaceutical composition of claim 178, wherein said disorder is nerveroot injury.
 199. The pharmaceutical composition of claim 178, whereinsaid nerve disorder is spinal cord compression.